( A ) Representative K562 locus comparing Lamin B2 pA - DamID z - scores in cycle sorted with replication timing . Arrows point to regions with increased mid - S signal compared to G1 G2 , which tend to overlap with regions that exhibit intermediate replication timing .

O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative K562 locus comparing Lamin B2 pA-DamID z-scores in cell cycle sorted cells with replication timing. Arrows point to regions with increased mid-S signal compared to G1 and G2, which tend to overlap with regions that exhibit intermediate replication timing.


( B ) Difference tracks in pA - DamID z - scores between cycle stages Data were smoothed by a running mean of 5

O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O

(B) Difference tracks in pA-DamID z-scores between the cell cycle stages Data were smoothed by a running mean of 5 bins


( C ) Scatterplots of Repli - seq vs . difference in smoothed pA - DamID z - scores between mid - S sorted either G1 or G2 sorted for K562 HCT116 ( two replicates ) . Bins with pA - DamID differences between G1 G2 bigger than 0 . 2 were filtered out to prevent artifacts from cycle dynamics . Blue shows a fitted loess curve . Spearman correlation coefficient ( ρ ) between absolute Repli - seq signal pA - DamID difference was used as a measure of enriched signal at mid - S replicating ( with a Repli - seq score around zero ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

(C) Scatterplots of Repli-seq vs. the difference in smoothed pA-DamID z-scores between mid-S sorted cells and either G1 or G2 sorted cells for K562 and HCT116 cells (two replicates). Bins with pA-DamID differences between G1 and G2 bigger than 0.2 were filtered out to prevent artifacts from cell cycle dynamics. Blue line shows a fitted loess curve. The Spearman correlation coefficient (ρ) between the absolute Repli-seq signal and pA-DamID difference was used as a measure of the enriched signal at mid-S replicating DNA (with a Repli-seq score around zero).


( D ) Scatterplots of changes in pA - DamID signal versus pA - DamID signal in G1 ( left - panels ) or G2 ( right - panels )

O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

(D) Scatterplots of changes in pA-DamID signal versus the pA-DamID signal in G1 (left-hand panels) or G2 cells (right-hand panels)


B C10M reduces binding of pCRP to immobilized PC . pCRP was incubated with p - aminophenyl phosphoryl choline agarose beads under binding conditions with without C10M . Porous solid column chromatography was then used to evaluate binding capacity of pCRP to PC . Biological replicates , n = 3 , mean ± . P value was calculated with Student ' s t - .

O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

B C10M reduces binding of pCRP to immobilized PC. pCRP was incubated with p-aminophenyl phosphoryl choline agarose beads under binding conditions with and without C10M. Porous solid column chromatography was then used to evaluate the binding capacity of pCRP to PC. Biological replicates, n=3, mean ± SEM. P value was calculated with Student's t-test.


A Flow cytometry was used to determine binding of pCRP to activated platelets . P - selectin positive platelets were analyzed for CRP - Atto 594 . Platelets were incubated without labeled pCRP ( control ) , with labeled pCRP ( 50 μg / ml ) ± C10M ( 1 : 100 molar ) . Results are displayed as mean ± . P values were calculated with ANOVA Tukey ' s post hoc . Biological replicate , n = 3 .

O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_TYPE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Flow cytometry was used to determine binding of pCRP to activated platelets. P-selectin positive platelets were analyzed for CRP-Atto 594 fluorescence. Platelets were incubated without labeled pCRP (control), and with labeled pCRP (50 μg/ml) ± C10M (1:100 molar ratio). Results are displayed as mean ± SEM. P values were calculated with ANOVA and Tukey's post hoc test. Biological replicate, n=3.


B Confocal microscopy of ADP - activated human platelets in presence of pCRP . Depicted are 3D reconstructions of monolayers of ADP ( 20 μM ) activated platelets . Washed platelets were detected by anti - CD62P ( green ) . 50 μg / ml pCRP - Atto 594 ( red ) was incubated with isolated platelets with ( bottom ) or without ( top ) C10M . pCRP localizes on platelets , which can be inhibited by C10M .

O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-ORGANISM B-CELL_TYPE O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O

B Confocal fluorescence microscopy of ADP-activated human platelets in the presence of pCRP. Depicted are 3D reconstructions of monolayers of ADP (20 μM) activated platelets. Washed platelets were detected by anti-CD62P (green). 50 μg/ml pCRP-Atto 594 (red) was incubated with isolated platelets with (bottom) or without (top) C10M. pCRP localizes on platelets, which can be inhibited by C10M.


C Western blot of pCRP binding to activated platelets . Platelets bind less pCRP when incubated with C10M as detected with anti - CRP antibody , compared to anti - GAPDH antibody as a control . Washed human platelets were incubated with pCRP pCRP + C10M , respectively , lysed separated on SDS - PAGE . D Densitometric of bands of Western blots ( biological replicates , n = 3 ) from experiment described in ( C ) . P value was calculated by Student ' s t - . Graph shows mean ± .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O B-CELL_TYPE O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Western blot of pCRP binding to activated platelets. Platelets bind less pCRP when incubated with C10M as detected with the anti-CRP antibody, compared to the anti-GAPDH antibody as a control. Washed human platelets were incubated with pCRP and pCRP+C10M, respectively, lysed and separated on SDS-PAGE. D Densitometric quantification of protein bands of Western blots (biological replicates, n=3) from the experiment described in (C). P value was calculated by Student's t-test. Graph shows mean ± SEM.


A CRP - coated platelets visualized by confocal microscopy bound to whole blood leukocytes . Washed platelets ( green ) were incubated in serum supplemented with 50 μg / ml pCRP - Atto 594 ( red ) , without ( left ) with C10M ( right , 1 : 100 molar ) . Platelets were then added to whole blood samples imaged after RBC lysis . Shown are platelet - leukocyte - with without CRP attached .

O B-GENEPROD O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE B-CELL_TYPE O O B-CELL_TYPE O O O O O O B-TISSUE O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE O O O B-GENEPROD O O

A CRP-coated platelets visualized by confocal microscopy bound to whole blood leukocytes. Washed platelets (green) were incubated in serum supplemented with 50 μg/ml pCRP-Atto 594 (red), without (left) and with C10M (right, 1:100 molar ratio). Platelets were then added to whole blood samples and imaged after RBC lysis. Shown are platelet-leukocyte-aggregates with and without CRP attached.


B , C CRP induces platelet - leukocyte aggregation in a time - dependent conformation - specific manner . Whole blood samples stimulated with either 50 μg / ml pCRP ( B ) or mCRP ( C ) were analyzed by flow cytometry . Double positive events ( CD62P CD14 ) were identified as platelet - monocyte complexes . Incubation was stopped at indicated time points . Displayed are mean ± . P values were calculated with ANOVA Tukey ' s post hoc . Biological replicates , n = 6 .

O O O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B,C CRP induces platelet-leukocyte aggregation in a time-dependent and conformation-specific manner. Whole blood samples stimulated with either 50 μg/ml pCRP (B) or mCRP (C) were analyzed by flow cytometry. Double positive events (CD62P and CD14) were identified as platelet-monocyte complexes. Incubation was stopped at indicated time points. Displayed are mean ± SEM. P values were calculated with ANOVA and Tukey's post hoc test. Biological replicates, n=6.


D Static adhesion of monocytes to 20 μg / ml fibrinogen was tested as described before . Purified monocytes ( 3 x 106 per ml ) were stimulated with 50 μg / ml pCRP 20 μM ADP allowed to adhere for 35 min . ADP was used to activate supplemented platelets . Monocyte adhesion was quantified by calorimetric method absorbance measured after 30 min .

O O B-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O B-CELL_TYPE B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

D Static adhesion of monocytes to 20 μg/ml fibrinogen was tested as described before. Purified monocytes (3 x 106 per ml) were stimulated with 50 μg/ml pCRP and 20 μM ADP and allowed to adhere for 35 min. ADP was used to activate supplemented platelets. Monocyte adhesion was quantified by calorimetric method and absorbance measured after 30 min.


E Expression of TNF , IL1β IL6 ( from left to right ) in monocytes analyzed by flow cytometry . Addition of C10M to whole blood samples inhibits CRP - dependent expression of pro - inflammatory cytokines . P values were calculated with ANOVA Tukey ' s post - hoc . Biological replicates , n = 6 ( IL6 ) , n = 5 ( TNF ) n = 4 ( IL1β ) , respectively ,

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O B-TISSUE I-TISSUE O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O

E Expression of TNF, IL1β and IL6 (from left to right) in monocytes analyzed by flow cytometry. Addition of C10M to the whole blood samples inhibits the CRP-dependent expression of pro-inflammatory cytokines. P values were calculated with ANOVA and Tukey's post-hoc test. Biological replicates, n=6 (IL6), n=5 (TNF) and n=4 (IL1β), respectively,


F Representative examples of whole blood samples treated with pCRP C10M visualized by confocal microscopy . Depicted are 3D reconstructions multiple focal planes at 20x magnification . leukocytes were stained with DAPI ( blue ) anti - TNF - FITC ( green ) .

O O O O B-TISSUE I-TISSUE O O O B-GENEPROD B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O

F Representative examples of whole blood samples treated with pCRP and C10M visualized by confocal fluorescence microscopy. Depicted are 3D reconstructions multiple focal planes at 20x magnification. The leukocytes were stained with DAPI (blue) and anti-TNF-FITC (green).


A Confocal microscopy of ADP - activated platelets bound to HUVEC mono layers . pCRP - Atto 594 ( depicted in red ) was added incubated with without C10M . Anti - CD62P - FITC antibody was used to detect platelets ( green ) . HUVEC nuclei were counterstained with DAPI ( blue ) . pCRP colocalizes with platelets on endothelial . C10M inhibits CRP binding to activated platelets . Scale bar 50 µm .

O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_TYPE O O B-CELL_LINE O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O O O O O O

A Confocal fluorescence microscopy of ADP-activated platelets bound to HUVEC mono cell layers. pCRP-Atto 594 (depicted in red) was added and incubated with and without C10M. Anti-CD62P-FITC antibody was used to detect the platelets (green). HUVEC nuclei were counterstained with DAPI (blue). pCRP colocalizes with platelets on the endothelial cells. C10M inhibits CRP binding to activated platelets. Scale bar 50 µm.


B , C of ICAM - 1 ( B ) VCAM - 1 ( C ) expression on pCRP * / mCRP - activated HUVECs . ICAM - 1 VCAM - 1 expression were measured by flow cytometry . ADP - stimulated platelets were added to each sample ( except for " Control " " pCRP " ) served as activated membranes for pCRP dissociation to pCRP * / mCRP . C10M inhibits generation of pCRP * / mCRP thereby reduces expression of ICAM - 1 VCAM - 1 . Mean ( MFI ) results in flow cytometry are shown with results normalized to control ,

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

B,C Quantification of ICAM-1 (B) and VCAM-1 (C) expression on pCRP*/mCRP-activated HUVECs. ICAM-1 and VCAM-1 expression were measured by flow cytometry. ADP-stimulated platelets were added to each sample (except for "Control" and "pCRP") and served as activated cell membranes for pCRP dissociation to pCRP*/mCRP. C10M inhibits the generation of pCRP*/mCRP and thereby reduces the expression of ICAM-1 and VCAM-1. Mean fluorescence intensity (MFI) results in flow cytometry are shown with results normalized to control,


D , E Expression of integrin subunit αM ( CD11b ) in neutrophils ( D ) CD14 + monocytes ( E ) was accessed by flow cytometry as described previously ( Kiefer et al , 2021 ) . Human whole blood was incubated with 25 µg / ml pCRP , 20 µM ADP C10M ( molar 1 : 100 , pCRP : C10M ) , respectively . CD11b expression was analyzed by flow cytometry in neutrophils ( CD16 + , SSC high ) monocytes ( CD14 + , SSC low ) . Shown are scatter plots of MFI results in flow cytometry with results normalized to control ,

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

D,E Expression of integrin subunit αM (CD11b) in neutrophils (D) and CD14+ monocytes (E) was accessed by flow cytometry as described previously (Kiefer et al, 2021). Human whole blood was incubated with 25 µg/ml pCRP, 20 µM ADP and C10M (molar ratio 1:100, pCRP:C10M), respectively. CD11b expression was analyzed by flow cytometry in neutrophils (CD16+, SSC high) and monocytes (CD14+, SSC low). Shown are scatter plots of MFI results in flow cytometry with results normalized to control,


F , G ROS generation in whole blood detected in CD14 + monocytes ( F ) neutrophils ( G ) by redox - indicator dihydroethidium ( DHE ; 10 µg / ml ) . Blood samples incubated for 3 hours at 37 ° C , 5 % CO2 with 50 µg / ml pCRP mCRP , 20 µM ADP C10M ( molar 1 : 100 , pCRP : C10M ) , respectively . Control was left unstimulated mCRP served as positive control ( Thiele et al , 2018 ) . were washed after red blood lysis analyzed by flow cytometry . Shown are MFI results with results normalized to control , mean ± . P values were calculated with ANOVA Tukey ' s post - hoc . Biological replicates , n = 6 .

O O O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE O O B-GENEPROD O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F,G ROS generation in whole blood detected in CD14+ monocytes (F) and neutrophils (G) by redox-indicator dihydroethidium (DHE; 10 µg/ml). Blood samples incubated for 3 hours at 37 °C, 5% CO2 with 50 µg/ml pCRP and mCRP, 20 µM ADP and C10M (molar ratio 1:100, pCRP:C10M), respectively. Control was left unstimulated and mCRP served as positive control (Thiele et al, 2018). Cells were washed after red blood cell lysis and analyzed by flow cytometry. Shown are MFI results with results normalized to control, mean ± SEM. P values were calculated with ANOVA and Tukey's post-hoc test. Biological replicates, n=6.


H pCRP * / mCRP dependent NETosis in isolated human neutrophils detected by confocal immunofluorescence microscopy . Isolated neutrophils incubated for 3 hours at 37 ° C , 5 % CO2 with 100 µg / ml pCRP with without PC : LPC liposomes ( LP ) C10M ( molar 1 : 100 , pCRP : C10M ) , respectively . Control was left unstimulated 100 nM phorbol 12 - myristate 13 - acetate ( PMA ) served as positive control . were washed , fixed stained , analyzed by confocal microscopy . Results are given as a of NETing / per ROI ,

O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

H pCRP*/mCRP dependent NETosis in isolated human neutrophils detected by confocal immunofluorescence microscopy. Isolated neutrophils incubated for 3 hours at 37 °C, 5% CO2 with 100 µg/ml pCRP with and without PC:LPC liposomes (LP) and C10M (molar ratio 1:100, pCRP:C10M), respectively. Control was left unstimulated and 100 nM phorbol 12-myristate 13-acetate (PMA) served as positive control. Cells were washed, fixed and stained, and analyzed by confocal microscopy. Results are given as a ratio of NETing cells/all cells per ROI,


B Detection of C10M by mass spectrometry in rat urine . Renal excretion of C10M detected by mass spectrometry in three rats ( biological replicates , n = 3 ) intravenously injected with C10M . Urine samples were collected at indicated timepoints . 80 % of applied C10M mass was excreted after 90

O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-TISSUE O B-TISSUE O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

B Detection of C10M by mass spectrometry in rat urine. Renal excretion of C10M detected by mass spectrometry in three rats (biological replicates, n=3) intravenously injected with C10M. Urine samples were collected at the indicated timepoints. 80% of the applied C10M mass was excreted after 90 min


C Immunohistochemistry of rat kidneys subjected to IRI i . p . pCRP application revealed distinct by anti - pCRP * / mCRP - 9C9 antibody ( green , arrows ) . C10M reduces deposition of total CRP in impaired tissue . No deposits in non - ischemic tissue ( sham ) . Exemplary stainings out of at least three are shown .

O B-EXP_ASSAY O B-ORGANISM B-TISSUE O O B-DISEASE O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O B-DISEASE O O O O O O B-EXP_ASSAY O O O O O O O O

C Immunohistochemistry of rat kidneys subjected to IRI and i.p. pCRP application revealed distinct staining by anti-pCRP*/mCRP-9C9 antibody (green, arrows). C10M reduces the deposition of total CRP in the impaired tissue. No deposits in the non-ischemic tissue (sham). Exemplary stainings out of at least three are shown.


D Tissue lysates of rat kidney were separated on SDS - PAGE total CRP was identified with anti - CRP antibody . A band at of mCRP ( ~ 23 kDa ) was detected in kidneys subjected to IRI pCRP , but not in treated additionally with C10M . household gene GAPDH served as a control for loading equal amounts of . 50 100 ng human pCRP , respectively , served as a positive control . Representative results are shown for replicated assays ( n = 3 ) .

O O O O B-ORGANISM B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O B-DISEASE B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

D Tissue lysates of rat kidney were separated on SDS-PAGE and total CRP was identified with anti-CRP antibody. A band at the size of mCRP (~23 kDa) was detected in kidneys subjected to IRI and pCRP, but not in animals treated additionally with C10M. The household gene protein GAPDH served as a control for loading equal amounts of protein. 50 and 100 ng human pCRP, respectively, served as a positive control. Representative results are shown for replicated assays (n=3).


E Renal excretion is impaired by pCRP * / mCRP - driven tissue damage . Blood urea nitrogen ( BUN ) was utilized as surrogate marker for excretion function of kidney . Blood samples were taken before surgical procedure ( preoperative ) 24 hours after procedure ( harvest ) . Graph shows mean ± .

O B-TISSUE O O O O B-GENEPROD O O B-GENEPROD O O O O O B-TISSUE B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O

E Renal excretion is impaired by pCRP*/mCRP-driven tissue damage. Blood urea nitrogen (BUN) was utilized as surrogate marker for the excretion function of the kidney. Blood samples were taken before the surgical procedure (preoperative) and 24 hours after the procedure (harvest). Graph shows mean ± SEM.


F Immunohistochemical detection of transmigrated CD68 + in IRI kidneys . of immunohistochemical results is shown as mean ± . pCRP ( 25 µg / ml ) increased number of CD68 + transmigrated into injured renal tissue significantly , while C10M abolished these effects . Presented are mean per ROI in each .

O B-EXP_ASSAY O O O B-GENEPROD O O B-DISEASE B-TISSUE O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

F Immunohistochemical detection of transmigrated CD68+ cells in IRI kidneys. Quantification of immunohistochemical results is shown as mean ± SEM. pCRP (25 µg/ml) increased the number of CD68+ cells transmigrated into injured renal tissue significantly, while C10M abolished these effects. Presented are mean cell counts per ROI in each animal.


G Periodic acid - Schiff ( PAS ) stained kidney sections show increased damage after renal IRI in rats when pCRP ( 25 µg / ml ) was injected i . v . tubulointerstitial injury was quantified by loss of tubular brush border by cast formation following an established protocol ( Megyesi et al , 1998 , 2001 ) . of immunohistochemical results is shown as mean ± .

O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY B-TISSUE O O O O O B-TISSUE B-DISEASE O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

G Periodic acid-Schiff (PAS) stained kidney sections show increased damage after renal IRI in rats when pCRP (25 µg/ml) was injected i.v. The tubulointerstitial injury was quantified by the loss of tubular brush border and by cast formation following an established protocol (Megyesi et al, 1998, 2001). Quantification of immunohistochemical results is shown as mean ± SEM.


H Representative results for immunohistochemical detection of transmigrated CD68 + in IRI kidneys . CD68 + are stained with HistoGreen substrate ( green ) . Scale bars indicate 100 µm .

O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-DISEASE B-TISSUE O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O

H Representative results for the immunohistochemical detection of transmigrated CD68+ cells in IRI kidneys. CD68+ cells are stained with HistoGreen substrate (green). Scale bars indicate 100 µm.


I Representative results for periodic acid - Schiff ( PAS ) stained kidney sections quantified in ( G ) . Scale bars indicate 100 µm .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY B-TISSUE O O O O O O O O O O O O O

I Representative results for the periodic acid-Schiff (PAS) stained kidney sections quantified in (G). Scale bars indicate 100 µm.


B Kaplan - Meier plots for control , pCRP , pCRP + C10M ( above ) C10M vs control ( bottom ) treatment for total allograft survival with without CRP ± C10M . Kaplan - Meier curves for different treatments were compared by Mantel - Cox log - rank . Allograft survival was significantly reduced by pCRP administration ( P = 0 . 0005 , median survival control vs pCRP , 7 . 8 vs 4 . 8 days ) . C10M masks CRP - accelerated hindlimb rejection ( median survival 7 . 2 days ) . Biological replicates , n = 4 per group .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-TISSUE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

B Kaplan-Meier plots for control, pCRP, and pCRP+C10M (above) and C10M vs control (bottom) treatment for total allograft survival with and without CRP ± C10M. Kaplan-Meier curves for different treatments were compared by Mantel-Cox log-rank test. Allograft survival was significantly reduced by pCRP administration (P=0.0005, median survival control vs pCRP, 7.8 vs 4.8 days). C10M masks the CRP-accelerated hindlimb rejection (median survival 7.2 days). Biological replicates, n=4 per group.


C Representative photographic examples of transplanted VCA hindlimb allografts . Shown are Lew hindlimb transplants in orthotopic situ on BN recipients three days after transplantation . Rats receiving i . p . pCRP presented VCA with massive edema ( left ) . C10M treatment inhibits pCRP - aggravated early graft rejection ( middle ) . depicted C10M control without CRP shows no clinical signs of rejection after five days ( right ) .

O O O O O O B-EXP_ASSAY B-TISSUE B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O B-EXP_ASSAY O O B-DISEASE O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O

C Representative photographic examples of transplanted VCA hindlimb allografts. Shown are Lew hindlimb transplants in orthotopic situ on BN recipients three days after transplantation. Rats receiving i.p. pCRP presented VCA with massive edema (left). C10M treatment inhibits pCRP-aggravated early graft rejection (middle). The depicted C10M control without CRP shows no clinical signs of rejection after five days (right).


D , E Representative immunofluorescence from transplanted grafts reveal distinct signals ( green ) using anti - CD68 anti - CRP antibodies , respectively . In C10M treated , less CD68 + infiltrate transplanted tissue . Scale bars , 100 μm . ( D ) Immunofluorescence of transplanted muscle ( E ) skin tissue revealed distinct CRP - deposition ( green ) in pCRP treated , but only minimal in rats treated additionally with C10M .

O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O B-TISSUE O O O B-TISSUE O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O

D,E Representative immunofluorescence images from transplanted grafts reveal distinct signals (green) using anti-CD68 and anti-CRP antibodies, respectively. In C10M treated animals, less CD68+ cells infiltrate the transplanted tissue. Scale bars, 100 μm. (D) Immunofluorescence of transplanted muscle and (E) skin tissue revealed distinct CRP-deposition (green) in pCRP treated animals, but only minimal staining in rats treated additionally with C10M.


F Exemplary tissue lysates of muscle skin probes separated on SDS - PAGE are depicted . Total human CRP was identified with an anti - CRP antibody .

O O O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM B-GENEPROD O O O O O O B-GENEPROD O O

F Exemplary tissue lysates of muscle and skin probes separated on SDS-PAGE are depicted. Total human CRP was identified with an anti-CRP antibody.


A Phagocytosis of pCRP - opsonized , heat - killed FITC - labeled S . pneumoniae by human monocytes serves as exemplary flow cytometry - phagocytosis . Bar chart shows phagocytic index ( of target positive of subtype / of subtype ) of un - opsonized ( control , - grey ) pCRP - opsonized targets ( red ) after 5 , 10 , 15 , 20 min , respectively . B Phagocytosis of pCRP - opsonized , heat - killed FITC - labeled E . coli by human monocytes serves as a second exemplary flow cytometry - phagocytosis . Bar chart shows phagocytic index as described above of un - opsonized ( control , - grey ) pCRP - opsonized targets ( red ) after 5 , 10 , 15 , 20 min , respectively . C Phagocytosis of pCRP - opsonized , heat - treated FITC - labeled zymosan by human monocytes serves as a third exemplary flow cytometry - phagocytosis . Bar chart shows phagocytic index as described above of un - opsonized ( control , - grey ) CRP - opsonized targets ( red ) after 5 , 10 , 15 , 20 min , respectively .

O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-ORGANISM B-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

A Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled S. pneumoniae by human monocytes serves as exemplary flow cytometry-based phagocytosis assay. Bar chart shows phagocytic index (percentage of target positive cells of subtype / all cells of subtype) of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. B Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled E. coli by human monocytes serves as a second exemplary flow cytometry-based phagocytosis assay. Bar chart shows phagocytic index as described above of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. C Phagocytosis of pCRP-opsonized, heat-treated and FITC-labeled zymosan by human monocytes serves as a third exemplary flow cytometry-based phagocytosis assay. Bar chart shows phagocytic index as described above of un-opsonized (control, dark-grey) and CRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively.


D Experiments described in ( A ) were repeated but with targets incubated with pCRP ( 100 µg / ml ) C10M ( 1 : 100 molar ) for 30 min , 37 ° C . Bar chart shows phagocytic index of S . pneumoniae - positive monocytes for un - opsonized ( control , - grey ) targets incubated with pCRP C10M ( green ) after 5 , 10 , 15 , 20 min , respectively . E Experiments described in ( B ) were repeated but with targets incubated with pCRP ( 100 µg / ml ) C10M ( 1 : 100 molar ) for 30 min , 37 ° C . Bar chart shows phagocytic index of E . coli - positive monocytes for un - opsonized ( control , - grey ) targets incubated with pCRP C10M ( green ) after 5 , 10 , 15 , 20 min , respectively . F Experiments described in ( C ) were repeated but with targets incubated with pCRP ( 100 µg / ml ) C10M ( 1 : 100 molar ) for 30 min , 37 ° C . Bar chart shows phagocytic index of zymosan - positive monocytes for un - opsonized ( control , - grey ) targets incubated with pCRP C10M ( green ) after 5 , 10 , 15 , 20 min , respectively .

O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O

D Experiments described in (A) were repeated but with targets incubated with pCRP (100 µg/ml) and C10M (1:100 molar ratio) for 30 min, 37 °C. Bar chart shows phagocytic index of S. pneumoniae-positive monocytes for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. E Experiments described in (B) were repeated but with targets incubated with pCRP (100 µg/ml) and C10M (1:100 molar ratio) for 30 min, 37 °C. Bar chart shows phagocytic index of E. coli-positive monocytes for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. F Experiments described in (C) were repeated but with targets incubated with pCRP (100 µg/ml) and C10M (1:100 molar ratio) for 30 min, 37 °C. Bar chart shows phagocytic index of zymosan-positive monocytes for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively.


G Phagocytosis of pCRP - opsonized , heat - killed FITC - labeled S . pneumoniae by human neutrophils . same blood samples described in ( A ) were analyzed for phagocytic index of S . pneumoniae by human neutrophils by flow cytometry . Bar chart shows phagocytic index of un - opsonized ( control , - grey ) pCRP - opsonized targets ( red ) after 5 , 10 , 15 , 20 min , respectively . H Phagocytosis of pCRP - opsonized , heat - killed FITC - labeled E . coli by human neutrophils . same blood samples described in ( B ) were analyzed for phagocytic index of S . pneumoniae by human neutrophils by flow cytometry . Bar chart shows phagocytic index of un - opsonized ( control , - grey ) pCRP - opsonized targets ( red ) after 5 , 10 , 15 , 20 min , respectively . I Phagocytosis of pCRP - opsonized , heat - killed FITC - labeled zymosan by human neutrophils . same blood samples described in ( C ) were analyzed for phagocytic index of S . pneumoniae by human neutrophils . Bar chart shows phagocytic index of un - opsonized ( control , - grey ) pCRP - opsonized targets ( red ) after 5 , 10 , 15 , 20 min , respectively .

O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O O B-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O O B-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-ORGANISM B-CELL_TYPE O O B-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

G Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled S. pneumoniae by human neutrophils. The same blood samples described in (A) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. H Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled E. coli by human neutrophils. The same blood samples described in (B) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. I Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled zymosan by human neutrophils. The same blood samples described in (C) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils. Bar chart shows phagocytic index of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively.


J Phagocytosis of pCRP + C10M - treated S . pneumoniae by human neutrophils . Results from experiments described in ( D ) were analyzed for phagocytic index of S . pneumoniae by human neutrophils by flow cytometry . Bar chart shows phagocytic index of S . pneumoniae - positive neutrophils for un - opsonized ( control , - grey ) targets incubated with pCRP C10M ( green ) after 5 , 10 , 15 , 20 min , respectively . K Phagocytosis of pCRP + C10M - treated E . coli by human neutrophils . Results from experiments described in ( E ) were analyzed for phagocytic index of S . pneumoniae by human neutrophils by flow cytometry . Bar chart shows phagocytic index of E . coli - positive neutrophils for un - opsonized ( control , - grey ) targets incubated with pCRP C10M ( green ) after 5 , 10 , 15 , 20 min , respectively . L Phagocytosis of pCRP + C10M - treated zymosan by human neutrophils . Results from experiments described in ( F ) were analyzed for phagocytic index of S . pneumoniae by human neutrophils by flow cytometry . Bar chart shows phagocytic index of zymosan - positive neutrophils for un - opsonized ( control , - grey ) targets incubated with pCRP C10M ( green ) after 5 , 10 , 15 , 20 min , respectively .

O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O

J Phagocytosis of pCRP+C10M-treated S. pneumoniae by human neutrophils. Results from experiments described in (D) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of S. pneumoniae-positive neutrophils for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. K Phagocytosis of pCRP+C10M-treated E. coli by human neutrophils. Results from experiments described in (E) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of E. coli-positive neutrophils for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. L Phagocytosis of pCRP+C10M-treated zymosan by human neutrophils. Results from experiments described in (F) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of zymosan-positive neutrophils for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively.


B . Blots showing expression of hTauAT ( hTau , Mr ~ 67 kDa ) / or endogenous mouse Tau ( mTau , Mr ~ 45 - 55 kDa ) in hippocampus of 10 month old heterozygous ( + / - ) homozygous ( + / + ) hTauAT mice in comparison to matched control ( Ctrl ) detected by pan - Tau antibody K9JA . Actin at 42 kDa serves as loading control .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O O O

B. Blots showing expression of hTauAT (hTau, Mr ~67 kDa) and/or endogenous mouse Tau (mTau, Mr ~45-55 kDa) in the hippocampus of 10 month old heterozygous (+/-) and homozygous (+/+) hTauAT mice in comparison to age matched control (Ctrl) detected by pan-Tau antibody K9JA. Actin at 42 kDa serves as loading control.


C . of ( B ) . of hTau / mTau indicating a higher hTauAT - expression in homozygous mice ( ~ 1 . 8 ) compared to heterozygous mice ( ~ 1 . 0 ) . Each bar represents an average of n = 4 , error bars represent .

O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

C. Quantification of (B). Ratio of hTau / mTau indicating a higher hTauAT -expression in homozygous mice (~1.8) compared to heterozygous mice (~1.0). Each bar represents an average of n=4 animals, error bars represent SEM.


D . of mRNA in hippocampi of heterozygous homozygous hTauAT 16 month old mice ( n = 4 per group ) .

O O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O

D. Quantification of mRNA levels in hippocampi of heterozygous and homozygous hTauAT 16 month old mice (n = 4 per group).


E . Distribution of hTauAT visualized by human Tau specific antibody HT7 in hippocampal CA3 - region of hetero - homozygous hTauATmice at of 14 month . Notice mis - sorted hTauAT in bodies dendrites ( arrowheads ) of pyramidal neurons of CA3 region immunreactivity of axons ( mossy , asterisks ) . By contrast control shows no immunoreactivity .

O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O

E. Distribution of hTauAT visualized by the human Tau specific antibody HT7 in the hippocampal CA3-region of hetero- and homozygous hTauATmice at the age of 14 month. Notice the mis-sorted hTauAT in cell bodies and dendrites (arrowheads) of pyramidal neurons of the CA3 region and immunreactivity of the axons (mossy fibers, asterisks). By contrast the control shows no immunoreactivity.


F . Elevated hTauAT - expression causes Tau - pathology inside CA3 of hippocampus . Note increase in Tau phosphorylation probed with antibody against pT217 , a site upstream of repeat domain . Arrowheads point to areas with phosphorylated mislocalized Tau in somata of pyramidal neurons in CA3 .

O O O O O O O B-GENEPROD O O O B-TISSUE O B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O

F. Elevated hTauAT -expression causes Tau-pathology inside area CA3 of the hippocampus. Note the increase in Tau phosphorylation probed with the antibody against pT217, a site upstream of the repeat domain. Arrowheads point to areas with phosphorylated and mislocalized Tau in cell somata of pyramidal neurons in area CA3.


G . Tauopathy detected by antibody AT180 specific for phosphorylation sites pT231 + pSer235 ( arrowheads denote somato - dendritic mislocalized tau ) .

O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O

G. Tauopathy detected by antibody AT180 specific for phosphorylation sites pT231+pSer235 (arrowheads denote somato-dendritic mislocalized tau).


H . Antibody PHF - 1 ( phosphorylation sites pSer396 + pSer404 ) illustrates pathological phosphorylation of Tau due to hTauAT expression especially in stratum lucidum ( asterisks ) of CA3 pyramidal neuroncell bodies ( arrows ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE B-SUBCELLULAR O O O O

H. Antibody PHF-1 (phosphorylation sites pSer396+pSer404) illustrates pathological phosphorylation of Tau due to hTauAT expression especially in stratum lucidum (asterisks) of area CA3 and pyramidal neuroncell bodies (arrows).


I . Tau phosphorylation detected by antibody AT8 ( pS202 + pT205 ) in stratum pyramidale of CA3 ( arrowhead ) . Note that immunoreactivity increases with expression level .

O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O

I. Tau phosphorylation detected by antibody AT8 (pS202+pT205) in stratum pyramidale of area CA3 (arrowhead). Note that immunoreactivity increases with expression level.


A . Conformational change of Tau detected by ALZ - 50 antibody immunoreactivity in stratum lucidum ( asterisks ) of CA3 somata in stratum pyramidale ( arrowheads ) .

O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O O B-TISSUE B-SUBCELLULAR O B-TISSUE I-TISSUE O O O O

A. Conformational change of Tau detected by ALZ-50 antibody immunoreactivity in stratum lucidum (asterisks) of area CA3 and somata in stratum pyramidale (arrowheads).


B . Tau aggregation confirmed by Gallyas silver of NFTs bearing neurons in hetero - homozygous hTauAT mice at 14 month of compared to non - reactive control littermate mice . In homozygous hTauAT mice extent of neurofibrillary tangles ( NFTs ) visualized by Gallyas silver is enhanced compared to heterozygous hTauAT mice ( arrowheads ) . control shows no silver - reactive Tau .

O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O

B. Tau aggregation confirmed by Gallyas silver staining of NFTs bearing neurons in hetero- and homozygous hTauAT mice at 14 month of age compared to non-reactive control littermate mice. In homozygous hTauAT mice the extent of neurofibrillary tangles (NFTs) visualized by Gallyas silver staining is enhanced compared to heterozygous hTauAT mice (white arrowheads). The control shows no silver-reactive Tau aggregates.


C . Western blot using pan - Tau antibody K9JA indicates sarcosyl - insoluble Tau species of human hTauAT ( upper band ) mouse Tau ( mTau , lower band ) . Note enhanced Tau aggregation in homozygous hTauAT mice compared to heterozygous hTauAT mice control mice . D . of ( C ) . of hTauAT / mTau indicates a stronger aggregation in homozygous hTauAT mice ( ~ 1 . 4 ) compared to heterozygous hTauAT mice ( ~ 0 . 7 ( n = 4 , error bars represent ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O

C. Western blot analysis using the pan-Tau antibody K9JA indicates sarcosyl-insoluble Tau species of human hTauAT (upper band) and mouse Tau (mTau, lower band). Note the enhanced Tau aggregation in homozygous hTauAT mice compared to heterozygous hTauAT mice and control mice.D. Quantification of (C). The ratio of hTauAT /mTau indicates a stronger aggregation in the homozygous hTauAT mice (~1.4) compared to heterozygous hTauAT mice (~0.7 (n=4 animals, error bars represent SEM).


E . Electron micrograph of CA3 region of hippocampus illustrating typical synapse with a perforated postsynaptic density ( asterisks ) in a control at 12 months . ( ds ) dendriticspine , ( pt ) presynaptic terminal ( m ) mitochondrium . F . Example of a degenerating synaptic bouton ( arrow ) containing swollen synaptic vesicles in proximity to a large terminal with a perforated postsynaptic density ( asterisks ) in a transgenic at 13 months . G . Electron dense neuronal cytoplasm with nucleoplasm various vacuoles ( arrow ) detected in CA3pyramidal neurons ( arrow ) at 13 months in transgenic in proximity to normally appearing neurons ( asterisks ) , degenerating apical dendrite ( d ) . H . Neuropil of a transgenic ( 13 months ) with a degenerating neuritic profile ( arrow ) . I . Degenerating dendrite ( arrow ) in a transgenic with darkened cytoplasm abnormally distributed mitochondria ( arrow ) . J . Degenerating dendrite containing electron - dense whorling fragments ( arrow ) in proximity to normally appearing synapses ( synapse with a perforated postsynaptic density , asterisks ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O B-TISSUE O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O

E. Electron micrograph of CA3 region of the hippocampus illustrating typical synapse with a perforated postsynaptic density (asterisks) in a control animal at 12 months. (ds) dendriticspine, (pt) presynaptic terminal and (m) mitochondrium.F. Example of a degenerating synaptic bouton (arrow) containing swollen synaptic vesicles in proximity to a large terminal with a perforated postsynaptic density (asterisks) in a transgenic animal at 13 months.G. Electron dense neuronal cytoplasm with dark nucleoplasm and various vacuoles (arrow) detected in CA3pyramidal neurons (arrow) at 13 months in transgenic animals in proximity to normally appearing neurons (asterisks), degenerating apical dendrite (d).H. Neuropil of a transgenic animal (13 months) with a degenerating neuritic profile (arrow).I. Degenerating dendrite (arrow) in a transgenic animal with darkened cytoplasm and abnormally distributed mitochondria (arrow).J. Degenerating dendrite containing electron-dense whorling membrane fragments (arrow) in proximity to normally appearing synapses (synapse with a perforated postsynaptic density, asterisks).


A . Western blot with pan - Tau K9JA antibody to estimate expression of hTauAT endogenous Tau ( mTau ) . Slice homogenates from non - transgenic littermate ( lane 1 , Ctrl ) , heterozygouse hTauAT ( lane 2 , ( + / - ) ) or homozygouse hTauAT slices ( lane 3 , ( + / + ) ) were analyzed after three in culture . Actin served as a loading control . B . of hTauAT expression ( n = 3 - 6 slice homogenates ) . between hTauAT endogenous mouse Tau is shown .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O

A. Western blot analysis with pan-Tau K9JA antibody to estimate expression levels of hTauAT and endogenous Tau (mTau). Slice homogenates from non-transgenic littermate (lane 1, Ctrl), heterozygouse hTauAT (lane 2, (+/-)) or homozygouse hTauAT slices (lane 3, (+/+)) were analyzed after three weeks in culture. Actin served as a loading control.B. Quantification of hTauAT expression levels (n=3-6 slice homogenates). The ratio between hTauAT and endogenous mouse Tau is shown.


C . Immunohistochemistry with antibodies against human Tau ( TauY9 antibodies ; middle panel ) dendritic marker MAP2 ( left panel ) in slice cultures at DIV 10 . Stratum radiatum of CA3 is shown . Although MAP2 is apparent , no signal is detected by TauY9 antibody in control slices . In contrast , in hTauAT slices human Tau is detected in both dendrites axons ( see asterisk in merged image ) .

O O B-EXP_ASSAY O O O O B-GENEPROD O B-EXP_ASSAY O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O

C. Immunohistochemistry with antibodies against human Tau (TauY9 antibodies; middle panel) and dendritic marker MAP2 (left panel) in slice cultures at DIV 10. Stratum radiatum of area CA3 is shown. Although MAP2 staining is apparent, no signal is detected by the TauY9 antibody in control slices. In contrast, in hTauAT slices human Tau is detected in both dendrites and axons (see asterisk in merged image).


D . Immunohistochemistry against phosphorylated Tau at PHF1 - epitope in CA3 of a control ( upper lane ) or hTauAT slice ( bottom lane ) . PHF1 is slightly visible in some axons in control slices . In contrast , PHF1 - phosphorylated Tau is apparent in somata , dendrites axons of neurons in hTauAT slice cultures .

O O B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O O B-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O O O

D. Immunohistochemistry against phosphorylated Tau at the PHF1-epitope in area CA3 of a control (upper lane) or hTauAT slice (bottom lane). PHF1 staining is slightly visible in some axons in control slices. In contrast, PHF1-phosphorylated Tau is apparent in somata, dendrites and axons of neurons in hTauAT slice cultures.


E . Higher magnification image of apical dendrites in CA3 in a hTauAT slice after immunohistochemistry against MAP2 ( red ) Tau ( K9JA antibody , green ) . Tau is seen in dendritic spines ( asterisks ) in contrast to MAP2 , which is restricted to dendritic shaft .

O O O O O O O B-SUBCELLULAR O B-TISSUE O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O

E. Higher magnification image of apical dendrites in area CA3 in a hTauAT slice after immunohistochemistry against MAP2 (red) and Tau (K9JA antibody, green). Tau staining is seen in dendritic spines (asterisks) in contrast to MAP2, which is restricted to the dendritic shaft.


A . Slices ( DIV15 ) from control mice were loaded with Ca + + sensitive dye Fura - 2AM . Ratiometric are presented at baseline after application of high potassium . Note increase in intracellular Ca + + ( [ Ca + + ] i ) after KCl application . B . Slices ( DIV15 ) from hTauAT mice were loaded with Ca + + sensitive dye Fura - 2AM . Ratiometric at baseline after application of high potassium in CA3 are depicted . Note increase in [ Ca + + ] i ( false color legend ) under both conditions in hTauAT slices compared to controls .

O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

A. Slices (DIV15) from control mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images are presented at baseline and after application of high potassium. Note the increase in intracellular Ca++ ([Ca++]i) after KCl application.B. Slices (DIV15) from hTauAT mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images at baseline and after application of high potassium in area CA3 are depicted. Note the increase in [Ca++]i (false color legend) under both conditions in hTauAT slices compared to controls.


C . Electrophysiological example traces ( control = black ; hTauAT = red ) of excitatory postsynaptic field potentials ( fEPSP ) depicts examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale ( s . p . ) of CA3 in slices in which calcium imaging experiments were conducted . example depolarization evokes averaged calcium influxes into neurons depicted in calcium imaging graph . graph shows mean of [ Ca + + ] i changes in response to high KCl in stratum radiatum stratum pyramidale of CA3 in Fura2 - AM loaded hippocampal slices . After depolarization [ Ca + + ] i rises up to ~ 600 nM in hTauAT slices ( red trace , n = 6 slices , prepared from at least three ) , compared to ~ 300 nM in control slices ( black trace , n = 6 slices ; prepared from at least three ) . Note that even under resting conditions [ Ca + + ] i is elevated to ~ 120nM due to hTauAT expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-TISSUE I-TISSUE O O O O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

C. Electrophysiological example traces (control = black; hTauAT = red) of excitatory postsynaptic field potentials (fEPSP) depicts examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale (s.p.) of area CA3 in slices in which calcium imaging experiments were conducted. The example depolarization evokes averaged calcium influxes into neurons depicted in the calcium imaging graph. The graph shows the mean of [Ca++]i changes in response to high KCl in stratum radiatum and stratum pyramidale of area CA3 in Fura2-AM loaded hippocampal slices. After depolarization [Ca++]i rises up to ~600 nM in hTauAT slices (red trace, n=6 slices, prepared from at least three animals), compared to ~300 nM in control slices (black trace, n=6 slices; prepared from at least three animals). Note that even under resting conditions [Ca++]i is elevated to ~120nM due to hTauAT expression.


D . of maximum [ Ca + + ] i from ratiometric as depicted in ( A ) ( B ) after background subtraction selection of regions of interest ( 10 circles of fixed diameter ) in stratum radiatum border to stratum pyramidale of CA3 . Under resting conditions [ Ca + + ] i was elevated in transgenic slice cultures ( ~ 120 nM , n = 11 ) compared to controls ( ~ 80 nM , n = 8 ) . Slices were incubated with one of following channel blockers : Nifedipine ( NIF , VGCC ; Ctrl n = 8 ; A152T : n = 7 ) , APV ( NMDAR ; Ctrl n = 7 ; A152T n = 8 ) , ifenprodil ( IFEN , NMDAR ; Ctrl n = 8 ; A152T n = 7 ) , memantine ( MEM , NMDAR ; Ctrl / A152T n = 6 ) , 6 - cyano - 7 - nitroquinoxaline - 2 , 3 - dione ( CNQX ; AMPAR ; Ctrl n = 8 ; A152T n = 7 ) ) , tetrodotoxin ( TTX , VGNaC ; Ctrl n = 4 ; A152T n = 5 ) , tetanus neurotoxin ( TeNT , neurotransmitter release ; A152T n = 6 ) or in Ca + + free buffer ( Ctrl n = 10 ; A152T n = 7 ) . For each experiment slice preparations from at least three different mice were used . One - way ANOVA followed by Tukey ' s post - hoc ; Ctrl : F ( 7 / 51 ) . 3 , 533 ; p = 0 . 0036 ; A152T : F ( 8 / 55 ) = 5 , 230 ; p < 0 . 0001 . For an overview of drug concentrations see EV Table 1 . E . of maximum peak increase in intracellular calcium in same slices than in ( D ) after high KCl stimulation . One way ANOVA followed by Tukey ' s post - hoc Ctrl : F ( 7 / 45 ) = 4 , 080 ; p = 0 . 0015 ; A152T : F ( 8 / 53 ) = 7 , 580 ; p < 0 . 0001 ) . Error bars represent ; One - Way ANOVA * p < 0 . 05 ; * * p < 0 . 01 * * * p < 0 . 001 .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Quantification of the maximum [Ca++]i from ratiometric images as depicted in (A) and (B) after background subtraction and selection of regions of interest (10 circles of fixed diameter) in stratum radiatum border to stratum pyramidale of area CA3. Under resting conditions [Ca++]i was elevated in transgenic slice cultures (~120 nM, n=11) compared to controls (~80 nM, n=8). Slices were incubated with one of the following channel blockers: Nifedipine (NIF, VGCC; Ctrl n=8; A152T: n=7), APV (NMDAR; Ctrl n=7; A152T n=8), ifenprodil (IFEN, NMDAR; Ctrl n=8; A152T n=7), memantine (MEM, NMDAR; Ctrl/A152T n=6), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; AMPAR; Ctrl n=8; A152T n=7)), tetrodotoxin (TTX, VGNaC; Ctrl n=4; A152T n=5), tetanus neurotoxin (TeNT, neurotransmitter release; A152T n=6) or in Ca++ free buffer (Ctrl n=10; A152T n=7). For each experiment slice preparations from at least three different mice were used. One-way ANOVA followed by Tukey's post-hoc test; Ctrl: F(7/51) .3,533; p=0.0036; A152T: F(8/55)=5,230; p<0.0001.For an overview of drug concentrations see EV Table 1.E. Quantification of the maximum peak increase in intracellular calcium in the same slices than in (D) after high KCl stimulation. One way ANOVA followed by Tukey's post-hoc test Ctrl: F(7/45)=4,080; p=0.0015; A152T: F(8/53)=7,580; p<0.0001).Error bars represent SEM; One-Way ANOVA *p<0.05; **p<0.01 and ***p<0.001.


A . Glutamate content in culture medium ( [ Glu ] e ) from either hTauAT ( n = 10 culture inserts containing 6 slices each ) or control slices ( n = 5 - 12 culture inserts ) analyzed at DIV 5 , 10 , 20 25 . Glutamate content from medium cultured with hTauAT slices is shown in of control at each time point . Increased were observed between DIV 5 DIV 20 . At DIV 25 glutamate already declined to control . One - way ANOVA followed by Tukey ' s post - hoc * p < 0 . 05 ; * * p < 0 . 01 * * * p < 0 . 001 . B . Lactate dehydrogenase ( LDH ) release ( a measure of cytotoxicity ) analyzed in hTauAT ( n = 17 - 19 culture inserts containing 6 slices each ) or control slices ( n = 15 - 16 culture inserts ) at DIV 5 , 10 , 20 25 . LDH release of hTauAT slices is shown in of control at each time point . A pronounced increase ( ~ 25 % ) in LDH release was observed due to hTauAT expression starting at DIV 10 but not at DIV 5 One - way ANOVA : * p < 0 . 05 ; * * p < 0 . 01 * * * p < 0 . 001 .

O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Glutamate content in the culture medium ([Glu]e) from either hTauAT (n=10 culture inserts containing 6 slices each) or control slices (n=5-12 culture inserts) analyzed at DIV 5, 10, 20 and 25. Glutamate content from medium cultured with hTauAT slices is shown in percent of control at each time point. Increased levels were observed between DIV 5 and DIV 20. At DIV 25 glutamate levels already declined to control levels. One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01 ***p<0.001.B. Lactate dehydrogenase (LDH) release (a measure of cytotoxicity) analyzed in hTauAT (n=17-19 culture inserts containing 6 slices each) or control slices (n=15-16 culture inserts) at DIV 5, 10, 20 and 25. LDH release of hTauAT slices is shown in percent of control at each time point. A pronounced increase (~25%) in LDH release was observed due to hTauAT expression starting at DIV 10 but not at DIV 5 One-way ANOVA: *p<0.05; **p<0.01 and ***p<0.001.


C . Slice cultures from control ( left ) , hTauAT ( middle ) or hTauAT treated with ceftriaxone ( CEF ; 100 µM ; right ) stained against neuronal ( NeuN ) at DIV 30 . Representative neuronal layers are shown . hTauAT slices show neuronal loss in granular layer of dentate gyrus pyramidal layer in region of hilus in CA3 ( middle panel , red arrows ) . Treatment with ceftriaxone for 30 days prevents neuronal loss in DG CA3 . D . NeuN positive bodies were counted in DG within pyramidal layer in CA3 CA1 at DIV 30 in defined regions of interest ( ROI ) . number of neurons was reduced in hTauAT slices , both within DG CA3 , whereas number of neurons in CA1 was only slightly affected . Neuronal loss was largely prevented in cultures treated with ceftriaxone ( CEF ) in dentate gyrus CA3 ( n = 14 - 19 slices per group ; prepared from at least 6 ) . One - way ANOVA followed by Tukey ' s post - hoc * p < 0 . 05 ; * * p < 0 .

O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O B-TISSUE O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-TISSUE I-TISSUE O O O O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Slice cultures from control (left), hTauAT (middle) or hTauAT treated with ceftriaxone (CEF; 100 µM; right) stained against neuronal nuclear protein (NeuN) at DIV 30. Representative neuronal cell layers are shown. hTauAT slices show neuronal loss in the granular cell layer of the dentate gyrus and the pyramidal cell layer in the region of the hilus in area CA3 (middle panel, red arrows). Treatment with ceftriaxone for 30 days prevents neuronal loss in DG and area CA3.D. NeuN positive cell bodies were counted in the DG and within the pyramidal cell layer in area CA3 and CA1 at DIV 30 in defined regions of interest (ROI). The number of neurons was reduced in hTauAT slices, both within area DG and CA3, whereas the number of neurons in the CA1 was only slightly affected. Neuronal loss was largely prevented in cultures treated with ceftriaxone (CEF) in dentate gyrus and area CA3 (n=14-19 slices per group and area; prepared from at least 6 animals). One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01


A . Representative microphotograph of spines on dendrites from control littermate mice at of 12 months . Apical dendrites from CA3pyramidal neurons are visualized by Golgi . contrast of photomicrographs was inverted . B . Dendritic spines from heterozygous hTauATmice , seen by Golgi . C . of dendritic spine densities in control littermate hTauAT mice . Note that spine density in heterozygous hTauAT is increased ( control : n = 2 ; heterozygous hTauATn = 4 ; an average of 5 dendrites of CA3pyramidal neurons were analyzed per ; unpaired t - ) .

O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-ORGANISM O O O O O O B-SUBCELLULAR O B-TISSUE B-CELL_TYPE O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-TISSUE B-CELL_TYPE O O O O O O O O O

A. Representative microphotograph of spines on dendrites from control littermate mice at the age of 12 months. Apical dendrites from CA3pyramidal neurons are visualized by Golgi staining. The contrast of photomicrographs was inverted.B. Dendritic spines from heterozygous hTauATmice, seen by Golgi staining.C. Quantification of dendritic spine densities in control littermate and hTauAT mice. Note that the spine density in heterozygous hTauAT is increased (control littermates: n = 2 animals; heterozygous hTauATn = 4 animals; an average of 5 dendrites of CA3pyramidal neurons were analyzed per animal; unpaired t-test).


D . Input / output curve of field potential recording in mossy fiber pathway in acute slices of 12 ± 1 month old mice demonstrating significantly enhanced basal synaptic transmission ( Multiple repeat ANOVA , F ( 9 , 126 ) = 2 . 00 , p = 0 . 044 ) in CA3 in mice expressing hTauAT . Scale bars : 500 µV vertical bar 20 msec horizontal bar .

O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O

D. Input / output curve of field potential recording in the mossy fiber pathway in acute slices of 12±1 month old mice demonstrating significantly enhanced basal synaptic transmission (Multiple repeat ANOVA,F(9, 126)=2.00, p=0.044) in area CA3 in mice expressing hTauAT. Scale bars: 500 µV vertical bar and 20 msec horizontal bar.


E . Long - term potentiation in mossy fiber pathway demonstrates no differences in long - term plasticity between control ( black ) hTauAT expressing mice ( red ) .

O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-ORGANISM O O O O

E. Long-term potentiation in the mossy fiber pathway demonstrates no differences in long-term plasticity between control (black) and hTauAT expressing mice (red).


F . Input / output curve of field potential recording in stratum pyramidale ( s . p . ) of CA3 in organotypic hippocampal slices at DIV 30 . Somatic field potentials are increased in hTauAT compared to control . Scale bars : 800 µV vertical bar 20 msec horizontal bar .

O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Input / output curve of field potential recording in stratum pyramidale (s.p.) of area CA3 in organotypic hippocampal slices at DIV 30. Somatic field potentials are increased in hTauAT compared to control littermates. Scale bars: 800 µV vertical bar and 20 msec horizontal bar.


A . Example of electrophysiological fEPSP recording in stratum pyramidale of CA3 in a control slice ( DIV30 ) during wash - in of 50 µm picrotoxin . This leads to blockage of GABAergic transmission followed by epileptiform discharges ( burst ) occurring approximately after 3 min . zoom - in reveals that such an epileptiform discharge ( burst ) consists of repetitive firings . B . Similar example trace as in ( A ) recorded in a hTauAT expressing slice ( red ) demonstrating an increase in burst frequency as well as in firings per burst due to hTauAT expression . C . of bursts per minute in DIV 10 slices from control hTauAT slices demonstrating a ~ 2 - fold increase in hTauAT slices ( n = 8 slices per group ) . D . Firings per burst are increased ~ 1 . 5 - fold already at early time point DIV10 due to hTauAT expression . E . of burst frequency in slices at DIV 30 demonstrates progression of pathological effect beyond that observed at DIV 10 . Burst frequency is enhanced in hTauAT expressing slices ( n = 10 slices ) . If Ceftriaxone ( CEF ) is applied to hTauAT slices pathological burst frequency enhancement is partly prevented ( n = 5 slices ) . F . Firings per burst are strongly enhanced due to hTauAT expression in slices at DIV 30 . Ceftriaxone prevents this pathological phenotype if applied during entire period of cultivation ( n = 6 slices ) .

O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

A. Example of electrophysiological fEPSP recording in stratum pyramidale of area CA3 in a control slice (DIV30) during wash-in of 50 µm picrotoxin. This leads to blockage of GABAergic transmission followed by epileptiform discharges (burst) occurring approximately after 3 min. The zoom-in reveals that such an epileptiform discharge (burst) consists of repetitive firings.B. Similar example trace as in (A) recorded in a hTauAT expressing slice (red) demonstrating an increase in burst frequency as well as in firings per burst due to hTauAT expression.C. Quantification of bursts per minute in DIV 10 slices from control littermates and hTauAT slices demonstrating a ~2-fold increase in hTauAT slices (n=8 slices per group).D. Firings per burst are increased ~1.5-fold already at the early time point DIV10 due to hTauAT expression.E. Analysis of burst frequency in slices at DIV 30 demonstrates the progression of the pathological effect beyond that observed at DIV 10. Burst frequency is enhanced in hTauAT expressing slices (n = 10 slices). If Ceftriaxone (CEF) is applied to hTauAT slices the pathological burst frequency enhancement is partly prevented (n = 5 slices).F. Firings per burst are strongly enhanced due to hTauAT expression in slices at DIV 30. Ceftriaxone prevents this pathological phenotype if applied during the entire period of cultivation (n = 6 slices).


G . Example of a control littermate organotypic slice culture at DIV 24 stained against NeuN ( red ) pan - Tau ( K9JA antibody , green ) . Axonal projections ( green ) of granule ( red ) in dentate gyrus are depicted . H . Hippocampal slice culture expressing hTauAT stained against NeuN ( red ) pan - Tau ( green ) showing mossy fiber sprouting in hilar region , CA3 dentate gyrus ( arrows ) .

O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O

G. Example of a control littermate organotypic slice culture at DIV 24 stained against NeuN (red) and pan-Tau (K9JA antibody, green). Axonal projections (green) of granule cells (red) in the dentate gyrus are depicted.H. Hippocampal slice culture expressing hTauAT stained against NeuN (red) and pan-Tau (green) showing mossy fiber sprouting in the hilar region, CA3 and dentate gyrus (white arrows).


A . of glutamate concentrations in medium of control hTauAT cultures in presence of either 50 nM tetanus neurotoxin ( TeNT ) or 1 µM tetrodotoxin ( TTX ) at DIV5 . Under both conditions , glutamate in culture medium of hTauAT slices decreased to control ( n = 6 culture inserts containing 6 slices each / group condition ) . B . of extracellular glutamate concentrations in culture medium of either control or hTauAT slices at DIV 20 in presence or absence of 50 µM benzyloxy - aspartic acid ( TBOA ) from DIV1 - DIV20 demonstrating a possible contribution of astrocytic glutamate dysregulation to increased extracellular glutamate in hTauAT slices ( n = 8 - 9 culture inserts containing 6 slices each / group condition ) . One - Way ANOVA F ( 3 , 30 ) = 14 , 89 ; p < 0 .

O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Quantification of glutamate concentrations in the medium of control and hTauAT cultures in the presence of either 50 nM tetanus neurotoxin (TeNT) or 1 µM tetrodotoxin (TTX) at DIV5. Under both conditions, glutamate levels in the culture medium of hTauAT slices decreased to control levels (n=6 culture inserts containing 6 slices each / group and condition).B. Quantification of extracellular glutamate concentrations in culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV1-DIV20 demonstrating a possible contribution of astrocytic glutamate dysregulation to increased extracellular glutamate in hTauAT slices (n=8-9 culture inserts containing 6 slices each / group and condition). One-Way ANOVA F(3,30)=14,89; p<0.001


C . of LDH release into culture medium of either control or hTauAT slices at DIV 20 in presence or absence of 50 µM benzyloxy - aspartic acid ( TBOA ) from DIV 1 to DIV 20 ( n = 6 - 12 culture inserts containing 6 slices each / group condition ) . One - Way ANOVA followed by Tukey ' s post - hoc .

O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Quantification of LDH release into the culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV 1 to DIV 20 (n=6-12 culture inserts containing 6 slices each / group and condition). One-Way ANOVA followed by Tukey's post-hoc test.


D . of extracellular glutamate concentrations in either control or hTauAT cultures treated ( n = 7 - 8 culture inserts containing 6 slices each / group condition ) with ceftriaxone from DIV 1 to DIV 25 demonstrating preventative effect of CEF on increase in extracellular glutamate in hTauAT slices . One - Way ANOVA F ( 3 , 25 ) = 9 . 075 ; p = 0 . 0003 ) .

O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Quantification of extracellular glutamate concentrations in either control or hTauAT cultures treated (n=7-8 culture inserts containing 6 slices each / group and condition) with ceftriaxone from DIV 1 to DIV 25 demonstrating the preventative effect of CEF on the increase in extracellular glutamate in hTauAT slices. One-Way ANOVA F(3,25)=9.075; p=0.0003).


E . LDH release at DIV 25 is strongly reduced due to long term treatment with ceftriaxone in hTauAT slice cultures ( n = 8 - 10 culture inserts containing 6 slices each / group condition ) . One Way ANOVA F ( 3 , 32 ) = 7 . 685 ; p = 0 . 0005 ) .

O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. LDH release at DIV 25 is strongly reduced due to long term treatment with ceftriaxone in hTauAT slice cultures (n=8-10 culture inserts containing 6 slices each / group and condition). One Way ANOVA F(3,32)=7.685; p=0.0005).


F . Intracellular calcium rise after KCl stimulation is not distinguishable from control slices in hTauAT slices treated with ceftriaxone for 15 days ( n = 5 - 9 slices ; prepared from at least three ) . One way ANOVA F ( ( 3 , 25 ) = 7 . 317 ; p = 0 . 0011 ) .

O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Intracellular calcium rise after KCl stimulation is not distinguishable from control slices in hTauAT slices treated with ceftriaxone for 15 days (n=5-9 slices; prepared from at least three animals). One way ANOVA F((3,25)=7.317; p=0.0011).


A . Genes involved in innate immunity are significantly upregulated in nuo - 6 isp - 1 worms . Gene expression changes in mitochondrial mutants were determined by sequencing compared to wild - type N2 worms . Results represent per million ( CPM ) expressed as a of wild - type of six biological replicates per . Data information : Error bars indicate . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance was determined using a one - way ANOVA with Dunnett ' s multiple comparison

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Genes involved in innate immunity are significantly upregulated in nuo-6 and isp-1 worms. Gene expression changes in the mitochondrial mutants were determined by RNA sequencing and compared to wild-type N2 worms. Results represent counts per million (CPM) expressed as a percentage of wild-type of six biological replicates per strain. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison test


B . Upregulation of innate immunity genes is dependent on p38 - mediated innate immune signaling pathway including NSY - 1 , SEK - 1 , PMK - 1 ATF - 7 . Gene expression changes were examined by quantitative RT - PCR of three biological replicates per . Data information : Error bars indicate . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance was determined using a one - way ANOVA with Dunnett ' s multiple comparison

O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Upregulation of innate immunity genes is dependent on the p38-mediated innate immune signaling pathway including NSY-1, SEK-1, PMK-1 and ATF-7. Gene expression changes were examined by quantitative RT-PCR of three biological replicates per strain. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison test


C . Using a reporter for innate immunity gene T24B8 . 5 confirms that innate immunity genes are upregulated in long - lived mitochondrial mutants that components of p38 - mediated innate immune signaling pathway are required for their upregulation . Scale bar indicates 250 µM . Three biological replicates per were quantified . Data information : Error bars indicate . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance was determined using a two - way ANOVA with Bonferroni post -

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Using a fluorescent reporter strain for the innate immunity gene T24B8.5 confirms that innate immunity genes are upregulated in the long-lived mitochondrial mutants and that components of the p38-mediated innate immune signaling pathway are required for their upregulation. Scale bar indicates 250 µM. Three biological replicates per strain were quantified. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a two-way ANOVA with Bonferroni post-test


A - D . Resistance to pathogens was tested by exposing worms to Pseudomonas aeruginosa PA14 in a slow kill . Both nuo - 6 isp - 1 long - lived mitochondrial mutants show increased resistance compared to wild - type worms ( A ) . Disruption of components of p38 - mediated innate immune signaling pathway ( nsy - 1 , sek - 1 , pmk - 1 , atf - 7 ) markedly decreases resistance to PA14 in wild - type ( B ) , nuo - 6 ( C ) isp - 1 ( D ) worms . strains were tested in a single parallel experiment . Two biological replicates per were performed with a total of at least 175 per . Data information : * * * p < 0 . 001 . Statistical significance for survival plots was determined with log - rank . Significance is indicated between listed on top other strains . Data from panel A is repeated in panels B , C , D for direct comparison .

O O O O O O O O O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D. Resistance to bacterial pathogens was tested by exposing worms to Pseudomonas aeruginosa strain PA14 in a slow kill assay. Both nuo-6 and isp-1 long-lived mitochondrial mutants show increased resistance compared to wild-type worms (A). Disruption of components of the p38-mediated innate immune signaling pathway (nsy-1, sek-1, pmk-1, atf-7) markedly decreases resistance to PA14 in wild-type (B), nuo-6 (C) and isp-1 (D) worms. All strains were tested in a single parallel experiment. Two biological replicates per strain were performed with a total of at least 175 animals per strain. Data information: ***p<0.001. Statistical significance for survival plots was determined with the log-rank test. Significance is indicated between the strain listed on top and all other strains. Data from panel A is repeated in panels B, C, and D for direct comparison.


A - H . To examine role of p38 - mediated innate immune signaling pathway in long - lifespan of nuo - 6 isp - 1 mitochondrial mutants , we crossed long - lived mitochondrial mutants to worms with mutations in nsy - 1 , sek - 1 , pmk - 1 atf - 7 ( gof ) . In each case , mutation of genes involved in p38 - mediated innate immunity signaling markedly reduces lifespan of long - lived mitochondrial mutant but have little or no impact on wild - type lifespan . In panel G , we utilized pmk - 1 RNAi instead of pmk - 1 mutation because of close proximity of isp - 1 pmk - 1 on same chromosome . EV indicates empty vector . lifespan results for panels A , B , D , E , F , H were performed in a single parallel experiment . Results are from a minimum of three biological replicates per . Data information : Statistical significance for survival plots was determined with log - rank . p - values indicate significance between red purple lines . Control strains are shown in multiple panels for direct comparison .

O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-H. To examine the role of the p38-mediated innate immune signaling pathway in the long-lifespan of the nuo-6 and isp-1 mitochondrial mutants, we crossed the long-lived mitochondrial mutants to worms with mutations in nsy-1, sek-1, pmk-1 and atf-7(gof). In each case, mutation of genes involved in p38-mediated innate immunity signaling markedly reduces the lifespan of the long-lived mitochondrial mutant but have little or no impact on wild-type lifespan. In panel G, we utilized pmk-1 RNAi instead of the pmk-1 mutation because of the close proximity of isp-1 and pmk-1 on the same chromosome. EV indicates empty vector. The lifespan results for panels A, B, D, E, F, and H were performed in a single parallel experiment. Results are from a minimum of three biological replicates per strain. Data information: Statistical significance for survival plots was determined with the log-rank test. p-values indicate significance between red and purple lines. Control strains are shown in multiple panels for direct comparison.


A , B . To determine extent to which p38 - mediated innate immunity pathway is activated in nuo - 6 isp - 1 mutants , we measured of phosphorylated PMK - 1 / p38 to total PMK - 1 / p38 by Western blotting . Representative blots are shown ( A ) . There is no difference in of phosphorylated to total PMK - 1 / p38 in long - lived mitochondrial mutants compared to wild - type ( B ) . Eight biological replicates of wild - type worms nine of mitochondrial mutants were quantified . Data information : Error bars indicate . * * * p < 0 . 001 . NS = not significant . Statistical significance was assessed using a one - way ANOVA with Dunnett ' s multiple comparison

O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-ORGANISM O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,B. To determine the extent to which the p38-mediated innate immunity pathway is activated in nuo-6 and isp-1 mutants, we measured the ratio of phosphorylated PMK-1/p38 to total PMK-1/p38 by Western blotting. Representative blots are shown (A). There is no difference in the ratio of phosphorylated to total PMK-1/p38 in the long-lived mitochondrial mutants compared to wild-type (B). Eight biological replicates of wild-type worms and nine of mitochondrial mutants were quantified. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test


F . Constitutive activation of atfs - 1 is sufficient to decrease food consumption to same level as in long - lived mitochondrial mutants . Three biological replicates per were measured , each including at least three technical replicates . atfs - 1 ( lof ) allele is gk3094 , atfs - 1 ( gof ) allele is et17 . Data information : Error bars indicate . * * * p < 0 . 001 . NS = not significant . Statistical significance was assessed using a one - way ANOVA with Dunnett ' s multiple comparison

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Constitutive activation of atfs-1 is sufficient to decrease food consumption to the same level as in long-lived mitochondrial mutants. Three biological replicates per strain were measured, each including at least three technical replicates. atfs-1(lof) allele is gk3094, atfs-1(gof) allele is et17. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test


A . A deletion of daf - 16 completely abolishes increased resistance to pathogens in isp - 1 mutants . p - value indicates significance of difference between red purple lines . Three biological replicates per were performed . Data information : Statistical significance was assessed using log - rank .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. A deletion of daf-16 completely abolishes the increased resistance to bacterial pathogens in isp-1 mutants. p-value indicates the significance of difference between red and purple lines. Three biological replicates per strain were performed. Data information: Statistical significance was assessed using log-rank test.


B , C . While daf - 16 RNAi effectively decreases expression of DAF - 16 target genes ( B , sod - 3 , dod - 3 , mtl - 1 , ftn - 1 , icl - 1 , sodh - 1 ) , it does not affect expression of any of innate immunity genes ( C , T24B8 . 5 , K08D8 . 5 , F55G11 . 8 , clec - 65 , clec - 67 , dod - 22 , Y9C9A . 8 C32H11 . 4 ) . This indicates that DAF - 16 is not required for expression of innate immune signaling pathway target genes in isp - 1 mutants . daf - 16 expression was knocked down using RNAi beginning at L4 stage of parental generation . was isolated from six biological replicates at young adult stage of experimental generation . from six biological replicates was pooled for sequencing .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

B,C. While daf-16 RNAi effectively decreases the expression of DAF-16 target genes (B, sod-3, dod-3, mtl-1, ftn-1, icl-1, sodh-1), it does not affect the expression of any of the innate immunity genes (C, T24B8.5, K08D8.5, F55G11.8, clec-65, clec-67, dod-22, Y9C9A.8 and C32H11.4). This indicates that DAF-16 is not required for expression of innate immune signaling pathway target genes in isp-1 mutants. daf-16 expression was knocked down using RNAi beginning at the L4 stage of the parental generation. RNA was isolated from six biological replicates at the young adult stage of the experimental generation. RNA from the six biological replicates was pooled for RNA sequencing.