Figure 4 ( A ) CHO cells ( for mammalian Atg8s ) or cells of S . cerevisiae ( for ScAtg8 ) were incubated in starvation medium ( EBSS or SD ‐ N , respectively ) for different time periods as indicated before isolation of RNA by the Tri ‐ reagent as explained in Materials and methods . For each gene , data obtained from non ‐ starved cells were set to an arbitrary value of 1 , and results from the corresponding starved cells were normalized accordingly . Results represent the means ± s . d . of three separate experiments . ( B ) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1 . Lysates ( 100 μg ) obtained in Ripa buffer were run on 12 % SDS - PAGE and analyzed by Western blot , using anti ‐ GATE ‐ 16 or anti ‐ tubulin antibodies . ( C ) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h , or starved for 3 or 13 h . Lysates ( 10 μg ) obtained in Ripa buffer were incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer ( 25 mM Tris , pH 7 . 4 , 50 mM KCl ) at 30 ° C for 45 min , in the presence or absence of 1 mM DTT . The reaction was stopped by addition of sample buffer and boiling , after which the samples were resolved on 15 % SDS - PAGE and subsequently analyzed by Western blot , using anti ‐ His monoclonal antibodies . The experiment was repeated six times ; a representative blot is shown . ( D ) Left panel : HeLa cells transfected with Myc ‐ GATE ‐ 16 ‐ HA or with an empty vector as control were labeled with [ 35S ] methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer . Lysates were immunoprecipitated using anti ‐ Myc antibodies and the immunoprecipitates were resolved on 15 % SDS - PAGE . Middle panel : HeLa cells transfected with Myc ‐ GATE ‐ 16 ‐ HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [ 35S ] methionine for 10 min , immediate lysis , analysis by SDS - PAGE and quantification ( right panel ) using NIH image program . Values are presented as the average percentage of unprimed form out of the total GATE ‐ 16 . ( * ) in all sections of this figure indicates non ‐ cleaved His6 ‐ GATE ‐ 16 ‐ HA or Myc ‐ GATE ‐ 16 ‐ HA and ( * * ) indicates cleaved His6 ‐ GATE ‐ 16 or Myc ‐ GATE ‐ 16 .

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Figure 4(A) CHO cells (for mammalian Atg8s) or cells of S. cerevisiae (for ScAtg8) were incubated in starvation medium (EBSS or SD‐N, respectively) for different time periods as indicated before isolation of RNA by the Tri‐reagent as explained in Materials and methods. For each gene, data obtained from non‐starved cells were set to an arbitrary value of 1, and results from the corresponding starved cells were normalized accordingly. Results represent the means±s.d. of three separate experiments.(B) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1. Lysates (100 μg) obtained in Ripa buffer were run on 12% SDS-PAGE and analyzed by Western blot, using anti‐GATE‐16 or anti‐tubulin antibodies.(C) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h, or starved for 3 or 13 h. Lysates (10 μg) obtained in Ripa buffer were incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer (25 mM Tris, pH 7.4, 50 mM KCl) at 30°C for 45 min, in the presence or absence of 1 mM DTT. The reaction was stopped by addition of sample buffer and boiling, after which the samples were resolved on 15% SDS-PAGE and subsequently analyzed by Western blot, using anti‐His monoclonal antibodies. The experiment was repeated six times; a representative blot is shown.(D) Left panel: HeLa cells transfected with Myc‐GATE‐16‐HA or with an empty vector as control were labeled with [35S]methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer. Lysates were immunoprecipitated using anti‐Myc antibodies and the immunoprecipitates were resolved on 15% SDS-PAGE. Middle panel: HeLa cells transfected with Myc‐GATE‐16‐HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [35S]methionine for 10 min, immediate lysis, analysis by SDS-PAGE and quantification (right panel) using NIH image program. Values are presented as the average percentage of unprimed form out of the total GATE‐16. (*) in all sections of this figure indicates non‐cleaved His6‐GATE‐16‐HA or Myc‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16 or Myc‐GATE‐16.


Figure 5 ( A ) Cleavage activity was tested by incubation of recombinant His6 ‐ HsAtg4A ( 0 . 1 μg ) and His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer at 30 ° C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis , using anti ‐ His monoclonal antibodies . The resulting bands from three separate experiments were quantified with a densitometer using the Bio ‐ Rad Multi ‐ Analyst program and are presented as the average percentage of cleaved form out of the total GATE ‐ 16 . ( * ) indicates non ‐ cleaved His6 ‐ GATE ‐ 16 ‐ HA and ( * * ) indicates cleaved His6 ‐ GATE ‐ 16 . ( B ) His6 ‐ HsAtg4A was incubated in the presence of 200 μM DTT at 4 ° C for 10 min . Reduced His6 ‐ HsAtg4A ( 0 . 1 μg ) was then incubated in 50 KT ( to obtain 15 μM DTT ) with the indicated concentrations of H2O2 at 25 ° C for 5 min , after which recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) was added and incubation proceeded at 30 ° C for 45 min . Reaction mixtures from three separate experiments were analyzed and are presented as explained in ( A ) . ( C ) His6 ‐ HsAtg4A ( 0 . 1 μg ) was incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) after the following procedures : no treatment ( lane 1 ) ; pretreatment with 200 μM DTT at 25 ° C for 5 min ( lane 2 ) ; treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25 ° C for 5 min ( lane 3 ) ; and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT ( lane 4 ) . Reaction mixtures were analyzed by Western blot using anti ‐ His monoclonal antibodies .

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Figure 5(A) Cleavage activity was tested by incubation of recombinant His6‐HsAtg4A (0.1 μg) and His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis, using anti‐His monoclonal antibodies. The resulting bands from three separate experiments were quantified with a densitometer using the Bio‐Rad Multi‐Analyst program and are presented as the average percentage of cleaved form out of the total GATE‐16. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.(B) His6‐HsAtg4A was incubated in the presence of 200 μM DTT at 4°C for 10 min. Reduced His6‐HsAtg4A (0.1 μg) was then incubated in 50 KT (to obtain 15 μM DTT) with the indicated concentrations of H2O2 at 25°C for 5 min, after which recombinant His6‐GATE‐16‐HA (0.3 μg) was added and incubation proceeded at 30°C for 45 min. Reaction mixtures from three separate experiments were analyzed and are presented as explained in (A).(C) His6‐HsAtg4A (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) after the following procedures: no treatment (lane 1); pretreatment with 200 μM DTT at 25°C for 5 min (lane 2); treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25°C for 5 min (lane 3); and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT (lane 4). Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies.


Figure 7 ( A ) Recombinant His6 ‐ HsAtg4AWT , His6 ‐ HsAtg4AC77A or His6 ‐ HsAtg4AC81S ( 0 . 1 μg ) was incubated with His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer at 30 ° C for 45 min in the presence or absence of 1 mM DTT . Reaction mixtures were analyzed by Western blot using anti ‐ His monoclonal antibodies . ( * ) indicates non ‐ cleaved His6 ‐ GATE ‐ 16 ‐ HA and ( * * ) indicates cleaved His6 ‐ GATE ‐ 16 . ( B ) His6 ‐ HsAtg4AC81S ( 0 . 1 μg ) was incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) following the same procedure as detailed in Figure 5C . Reaction mixtures were analyzed and are presented as explained in ( A ) .

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Figure 7(A) Recombinant His6‐HsAtg4AWT, His6‐HsAtg4AC77A or His6‐HsAtg4AC81S (0.1 μg) was incubated with His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence or absence of 1 mM DTT. Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.(B) His6‐HsAtg4AC81S (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) following the same procedure as detailed in Figure 5C. Reaction mixtures were analyzed and are presented as explained in (A).


Figure 8 ( A ) CHO cells stably expressing GFP ‐ GATE ‐ 16 were transiently transfected with HsAtg4AWT ‐ Myc ‐ His6 or HsAtg4AC81S ‐ Myc ‐ His6 . At 24 h post ‐ transfection , cells were starved for 2 . 5 h after which they were fixed , permeabilized and incubated with anti ‐ Myc monoclonal antibodies . Upper panel : typical images of cells transfected with the above constructs , as visualized using a confocal microscope ; representative autophagosomes are indicated by arrows . Lower panel : quantification of the average number of autophagosomes per cell in the different transfectants . Images of the fixed cells were visualized using a Nikon eclipse TE300 fluorescent microscope and used for quantification of the number of autophagosomes per cell . The results presented are the means ± s . d . of a total of 100 cells from three separate experiments . ( * ) indicates significance at P0 . 001 . ( B ) Left panel : HEK 293 cells were cotransfected with GFP ‐ GATE ‐ 16 and each of the Atg4A constructs mentioned in ( A ) . At 40 h post ‐ transfection , the cells were starved for 2 . 5 h , lysed in Ripa buffer and 100 μg of each lysate was loaded to 10 % SDS - PAGE and subsequently analyzed with anti ‐ GFP antibodies to detect the transfected GATE ‐ 16 , anti ‐ Myc antibodies to detect the transfected HsAtg4A and anti ‐ tubulin antibodies as control . ( * ) indicates non ‐ lipidated GFP ‐ GATE ‐ 16 and ( * * ) indicates lipidated GFP ‐ GATE ‐ 16 . Right panel : results from three separate experiments , as detailed in the left panel , were analyzed using NIH image program and quantified as follows : the amount of lipidated GFP ‐ GATE ‐ 16 out of the total of GFP ‐ GATE ‐ 16 was calculated for each mutant in each experiment . The value obtained for HsAtg4AWT in each experiment was set to 100 % and the relative lipidation in cells transfected with the mutant was calculated accordingly .

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Figure 8(A) CHO cells stably expressing GFP‐GATE‐16 were transiently transfected with HsAtg4AWT‐Myc‐His6 or HsAtg4AC81S‐Myc‐His6. At 24 h post‐transfection, cells were starved for 2.5 h after which they were fixed, permeabilized and incubated with anti‐Myc monoclonal antibodies. Upper panel: typical images of cells transfected with the above constructs, as visualized using a confocal microscope; representative autophagosomes are indicated by arrows. Lower panel: quantification of the average number of autophagosomes per cell in the different transfectants. Images of the fixed cells were visualized using a Nikon eclipse TE300 fluorescent microscope and used for quantification of the number of autophagosomes per cell. The results presented are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001.(B) Left panel: HEK 293 cells were cotransfected with GFP‐GATE‐16 and each of the Atg4A constructs mentioned in (A). At 40 h post‐transfection, the cells were starved for 2.5 h, lysed in Ripa buffer and 100 μg of each lysate was loaded to 10% SDS-PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16, anti‐Myc antibodies to detect the transfected HsAtg4A and anti‐tubulin antibodies as control. (*) indicates non‐lipidated GFP‐GATE‐16 and (**) indicates lipidated GFP‐GATE‐16. Right panel: results from three separate experiments, as detailed in the left panel, were analyzed using NIH image program and quantified as follows: the amount of lipidated GFP‐GATE‐16 out of the total of GFP‐GATE‐16 was calculated for each mutant in each experiment. The value obtained for HsAtg4AWT in each experiment was set to 100% and the relative lipidation in cells transfected with the mutant was calculated accordingly.


Figure 9 ( A ) HeLa cells were transiently transfected with GFP - HsAtg4BWT or GFP ‐ HsAtg4BC78S . At 24 h post ‐ transfection , cells were starved for 2 . 5 h in the presence of 4 mM H2O2 after which they were fixed , permeabilized and incubated with anti ‐ LC3 polyclonal antibodies . Cells were visualized ( upper panel ) and quantified ( lower panel ) as explained in Figure 8 . Representative autophagosomes are indicated in the upper panel by arrows . The results presented in the lower panel are the means ± s . d . of a total of 100 cells from three separate experiments . ( * ) indicates significance at P0 . 001 . ( B ) Left panel : HeLa cells transfected and treated as in ( A ) were lysed in Ripa buffer and 40 μg of each lysate was loaded to 12 % SDS - PAGE and subsequently analyzed with anti ‐ LC3 antibodies and anti ‐ GFP antibodies to detect the transfected HsAtg4B and anti ‐ tubulin antibodies as control . ( * ) indicates non ‐ lipidated LC3 and ( * * ) indicates lipidated LC3 . Right panel : results from three separate experiments , as detailed in the left panel , were analyzed as explained in Figure 8B .

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Figure 9(A) HeLa cells were transiently transfected with GFP-HsAtg4BWT or GFP‐HsAtg4BC78S. At 24 h post‐transfection, cells were starved for 2.5 h in the presence of 4 mM H2O2 after which they were fixed, permeabilized and incubated with anti‐LC3 polyclonal antibodies. Cells were visualized (upper panel) and quantified (lower panel) as explained in Figure 8. Representative autophagosomes are indicated in the upper panel by arrows. The results presented in the lower panel are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001.(B) Left panel: HeLa cells transfected and treated as in (A) were lysed in Ripa buffer and 40 μg of each lysate was loaded to 12% SDS-PAGE and subsequently analyzed with anti‐LC3 antibodies and anti‐GFP antibodies to detect the transfected HsAtg4B and anti‐tubulin antibodies as control. (*) indicates non‐lipidated LC3 and (**) indicates lipidated LC3. Right panel: results from three separate experiments, as detailed in the left panel, were analyzed as explained in Figure 8B.


Figure 1D . Representative genotyping of the c . 1192C > A mutation by allele - specific PCR . For each sample , two PCRs were performed , one with a specific primer for the 1192C allele ( PCR 1 ) and a second with specific primers for the 1192A allele ( PCR 2 ) . An 821 - bp band indicated the presence of the allele ; amplification failure indicated the absence of the allele . The 434 bp amplification product of the hGH control primer is presented in all lanes . In the gel , the first lane ( MW ) shows the 100 bp DNA ladder marker ; for the other lanes , C and A indicate the product from PCR 1 and PCR 2 , respectively , amplified from RIF + / + ( 1 ( CC ) ) , RIF + / - ( 2 ( CA ) ) and RIF - / - ( 3 ( AA ) ) samples .

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Figure 1D. Representative genotyping of the c.1192C>A mutation by allele-specific PCR. For each sample, two PCRs were performed, one with a specific primer for the 1192C allele (PCR 1) and a second with specific primers for the 1192A allele (PCR 2). An 821-bp band indicated the presence of the allele; amplification failure indicated the absence of the allele. The 434 bp amplification product of the hGH control primer is presented in all lanes. In the gel, the first lane (MW) shows the 100 bp DNA ladder marker ; for the other lanes, C and A indicate the product from PCR 1 and PCR 2, respectively, amplified from RIF+/+ (1(CC)), RIF+/- (2(CA)) and RIF-/- (3(AA)) samples.


Figure 2A and B . Flow analysis of SLC44A2 expression at the surface of RBCs from control donors ( red ) , RIF - proband 1 ( blue ) , and VER - proband 2 ( green ) using anti - RIF ( A ) and anti - VER antibodies ( B ) . The light gray profile corresponds to RBCs incubated with only the secondary antibody .

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Figure 2A and B. Flow analysis of SLC44A2 expression at the surface of RBCs from control donors (red), RIF- proband 1 (blue), and VER- proband 2 (green) using anti-RIF (A) and anti-VER antibodies (B). The light gray profile corresponds to RBCs incubated with only the secondary antibody.


Figure 3B . Western blotting shows that the anti - RIF and anti - VER antibodies bind to HuSLC44A2 . Western blots of whole Sf9 lysates ( WCL ) expressing full - length recombinant human SLC44A2 cDNA ( rHuSLC44A2 ) were probed with RIF and VER antisera ( 1 : 10 ) . Lanes 1 and 2 show strong binding to rHuSLC44A2 . Titer differences in the sera required longer exposure ( 10 min ) of the filter to the film to develop the image with VER serum compared to only 30 second exposure for RIF serum . A similar blot of immunoprecipitated rHuSLC44A2 from the Sf9 lysates similarly reacted with RIF and VER sera ( lanes 3 and 4 ) showed equivalent exposure times ( 30 seconds for both sera ) when tested against the concentrated rHuSLC44A2 protein . C . The anti RIF and VER antibodies bound to immunoprecipitated wild - type SLC44A2 in lysates of the UM - SCC - 47 human squamous cancer cell line ( exposure time 5 min ) . D . Western blot of murine Slc44a2 protein immunoprecipitated from wild - type and Slc44a2 knockout mice with rabbit anti CTL2 - NT serum was probed with anti CTL2 - NT ( left panel ) and with VER serum ( center Panel ) or RIF serum ( right panel ) . The left panel was developed with goat anti rabbit IgG heavy and light chain ; the center and right panels were developed with rabbit anti human IgG and IgM heavy and light chain

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Figure 3B. Western blotting shows that the anti-RIF and anti-VER antibodies bind to HuSLC44A2. Western blots of whole Sf9 lysates (WCL) expressing full-length recombinant human SLC44A2 cDNA (rHuSLC44A2) were probed with RIF and VER antisera (1:10). Lanes 1 and 2 show strong binding to rHuSLC44A2. Titer differences in the sera required longer exposure (10 min) of the filter to the film to develop the image with VER serum compared to only 30 second exposure for RIF serum. A similar blot of immunoprecipitated rHuSLC44A2 from the Sf9 lysates similarly reacted with RIF and VER sera (lanes 3 and 4) showed equivalent exposure times (30 seconds for both sera) when tested against the concentrated rHuSLC44A2 protein.C. The anti RIF and VER antibodies bound to immunoprecipitated wild-type SLC44A2 in lysates of the UM-SCC-47 human squamous cancer cell line (exposure time 5 min).D. Western blot of murine Slc44a2 protein immunoprecipitated from wild-type and Slc44a2 knockout mice with rabbit anti CTL2-NT serum was probed with anti CTL2-NT (left panel) and with VER serum (center Panel) or RIF serum (right panel). The left panel was developed with goat anti rabbit IgG heavy and light chain ; the center and right panels were developed with rabbit anti human IgG and IgM heavy and light chain specific


Figure 4A . CT angiography of the giant unruptured intracranial aneurysm on the right posterior cerebellar artery ( PCA , blue arrow ) . B . 3D volume rendered of multiple cerebral aneurysms on the right PCA ( a ) , on right posterior communicating artery ( b ) , and on right middle cerebral artery ( c ) . C and D . Angiograms before and after embolization of the PCA aneurysm , respectively .

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Figure 4A. CT angiography of the giant unruptured intracranial aneurysm on the right posterior cerebellar artery (PCA, blue arrow).B. 3D volume rendered of multiple cerebral aneurysms on the right PCA (a), on right posterior communicating artery (b), and on right middle cerebral artery (c).C and D. Angiograms before and after embolization of the PCA aneurysm, respectively.


Figure 5A . Terminal differentiation of HSPCs from healthy control and SLC44A2null proband was monitored via α4 - integrin and band 3 levels of GPApos cells at day 6 after EPO addition . B . Cell proliferation during in vitro erythropoiesis .

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Figure 5A. Terminal differentiation of HSPCs from healthy control and SLC44A2null proband was monitored via α4-integrin and band 3 levels of GPApos cells at day 6 after EPO addition.B. Cell proliferation during in vitro erythropoiesis.


Figure 6A . SLC44A2 transcript variants P1 and P2 were amplified from cDNA samples isolated from control reticulocytes ( CTRL RETIC ) , neutrophils ( CTRL NEUTRO ) , and PBMC of the VER - proband 2 ( VER PBMC ) . The purity of reticulocytes and neutrophils was verified by flow cytometry using anti - CD71 and anti - CD16 antibodies , respectively . D . Effect of anti - VER and anti - RIF on the adhesion of neutrophils from healthy controls to endothelial cells ( HMEC - 1 ) . Adherent neutrophils were quantified and means ± standard error of the median of 3 independent experiments are shown . * * P < . 01 , * * * P < . 001 , * * * * P < . 0001 . Statistical analyses were performed using GraphPad Prism 7 . Statistical test was performed using paired non - parametric t - test ( Wilcoxon t - test ) .

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Figure 6A. SLC44A2 transcript variants P1 and P2 were amplified from cDNA samples isolated from control reticulocytes (CTRL RETIC), neutrophils (CTRL NEUTRO), and PBMC of the VER- proband 2 (VER PBMC). The purity of reticulocytes and neutrophils was verified by flow cytometry using anti-CD71 and anti-CD16 antibodies, respectively.D. Effect of anti-VER and anti-RIF on the adhesion of neutrophils from healthy controls to endothelial cells (HMEC-1). Adherent neutrophils were quantified and means ± standard error of the median of 3 independent experiments are shown. **P < .01, ***P < .001, ****P < .0001. Statistical analyses were performed using GraphPad Prism 7. Statistical test was performed using paired non-parametric t-test (Wilcoxon t-test).


Figure 1B 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 .

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Figure 1B 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.


Figure 2A 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 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 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 .

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Figure 2A 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 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 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.


Figure 3A 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 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 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 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 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 ) .

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Figure 3A 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 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 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 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 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).


Figure 4A 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 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 ) 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 ) 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 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 ,

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A 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 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) 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) 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 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,


Figure 5B 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 minC 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 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 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 + 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 . 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 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 the periodic acid - Schiff ( PAS ) stained kidney sections quantified in ( G ) . Scale bars indicate 100 µm .

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Figure 5B 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 minC 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 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 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+ 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. 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 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 the periodic acid-Schiff (PAS) stained kidney sections quantified in (G). Scale bars indicate 100 µm.


Figure 6B 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 ) . The depicted C10M control without CRP shows no clinical signs of rejection after five days ( right ) . 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 and skin probes separated on SDS - PAGE are depicted . Total human CRP was identified with an anti - CRP antibody .

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Figure 6B 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). The depicted C10M control without CRP shows no clinical signs of rejection after five days (right).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 and skin probes separated on SDS-PAGE are depicted. Total human CRP was identified with an anti-CRP antibody.


Figure 7A 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 ) 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A 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) 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 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 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.


Figure 1Left : Representative time - lapse images showing the migration of single hRPE cells on the surface of polyacrylamide gels with uniform stiffness ( top ) and stiffness gradient ( bottom ) , referring as random migration and durotaxis migration respectively . Yellow line is the cell outline and blue line is the cell migration trajectory . Scale bar : 50 μm . Middle : Angular displacement of hRPE cells on the uniform stiffness gels ( top ) and stiffness gradient gels ( bottom ) . Right : y / x FMI ( forward migration index ) , velocity and persistence of hRPE cells with random migration and durotaxis . Left : Representative immunofluorescent staining of F - actin in control and CK - 666 ( 100 μM , 3 h ) treated hRPEs . Scale bar : 20 μm . Right : Y FMI of control and CK - 666 ( 100 μM , pre - treated for 1h and incubated for the whole experiment ) treated hRPEs moving on stiffness gradient gel . Left : Representative immunofluorescent staining of α - tubulin in control and nocodazole ( 8 μM , 30 min ) treated hRPEs . Scale bar : 20 μm . Right : Y FMI of control and nocodazole ( 8 μM , pre - treated for 10 min and incubated for the whole experiment ) treated hRPEs moving on stiffness gradient gel .

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Figure 1Left: Representative time-lapse images showing the migration of single hRPE cells on the surface of polyacrylamide gels with uniform stiffness (top) and stiffness gradient (bottom), referring as random migration and durotaxis migration respectively. Yellow line is the cell outline and blue line is the cell migration trajectory. Scale bar: 50 μm. Middle: Angular displacement of hRPE cells on the uniform stiffness gels (top) and stiffness gradient gels (bottom). Right: y/x FMI (forward migration index), velocity and persistence of hRPE cells with random migration and durotaxis.Left: Representative immunofluorescent staining of F-actin in control and CK-666 (100 μM, 3 h) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and CK-666 (100 μM, pre-treated for 1h and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel.Left: Representative immunofluorescent staining of α-tubulin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and nocodazole (8 μM, pre-treated for 10 min and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel.


Figure 2Representative immunofluorescent staining of γ - tubulin ( yellow ) and DAPI ( blue ) in control and centrinone - B ( 500 nM , 7 days ) treated hRPEs . Angular displacement and y FMI of control and centrinone - B ( 500 nM , 7 days ) treated hRPEs moving on stiffness gradient gels . Western blot showing the protein level of CAMSAP2 in negative control and CAMSAP2 siRNA transfected hRPEs . GAPDH was used as loading control . Bar chart shows quantification of protein levels normalized to GAPDH in each condition . Angular displacement and y FMI of negative control and CAMSAP2 siRNA transfected hRPEs moving on stiffness gradient gels . Western blot showing the protein level of AKAP450 in negative control and AKAP450 siRNA transfected hRPEs . α - tubulin was used as loading control . Bar chart shows quantification of protein levels normalized to α - tubulin in each condition . Angular displacement and y FMI of control and AKAP450 siRNA transfected hRPEs moving on stiffness gradient gels . Left : Representative immunofluorescent staining of α - tubulin in control , CAMSAP2 siRNA transfected and centrinone - B ( 500 nM , 7 days ) treated hRPEs . Scale bar : 20 μm . Right : Quantification of microtubule fluorescence intensity . Western blot showing the protein level of α - tubulin in control , CAMSAP2 siRNA transfected and centrinone - B ( 500 nM , 7 days ) treated hRPEs . GAPDH is used as loading control . Bar chart shows quantification of protein levels normalized to GAPDH in each condition . Left : Representative live - cell fluorescence imaging of EGFP - EB1 in control , CAMSAP2 siRNA transfected and centrinone - B ( 500 nM , 7 days ) treated hRPEs . Scale bar : 20 μm . Right : Quantification of EB1 growth speed obtained by plusTipTracker . Vinlin plot center lines denote the median , and dashed lines represent the 25th and 75th percentiles .

O B-PANEL_START O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative immunofluorescent staining of γ-tubulin (yellow) and DAPI (blue) in control and centrinone-B (500 nM, 7 days) treated hRPEs.Angular displacement and y FMI of control and centrinone-B (500 nM, 7 days) treated hRPEs moving on stiffness gradient gels.Western blot showing the protein level of CAMSAP2 in negative control and CAMSAP2 siRNA transfected hRPEs. GAPDH was used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition.Angular displacement and y FMI of negative control and CAMSAP2 siRNA transfected hRPEs moving on stiffness gradient gels.Western blot showing the protein level of AKAP450 in negative control and AKAP450 siRNA transfected hRPEs. α-tubulin was used as loading control. Bar chart shows quantification of protein levels normalized to α-tubulin in each condition.Angular displacement and y FMI of control and AKAP450 siRNA transfected hRPEs moving on stiffness gradient gels.Left: Representative immunofluorescent staining of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of microtubule fluorescence intensity.Western blot showing the protein level of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition.Left: Representative live-cell fluorescence imaging of EGFP-EB1 in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of EB1 growth speed obtained by plusTipTracker. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.


Figure 3Western blot showing the protein level of active RhoA , total RhoA in negative control and CAMSAP2 siRNA transfected hRPEs . GAPDH is used as loading control . Bar chart shows quantification of protein levels normalized to GAPDH in each condition . Left : Representative live - cell fluorescence imaging of GFP - AHPH in negative control and CAMSAP2 siRNA transfected hRPEs . Scale bar : 20 μm . Right : Quantification of GFP - AHPH fluorescence intensity . Left : Representative immunofluorescent staining of pMLC ( yellow ) and DAPI ( blue ) in negative control and CAMSAP2 siRNA transfected hRPEs . Scale bar : 20 μm . Right : Quantification of pMLC fluorescence intensity . Below : Western blot showing the protein level of pMLC in negative control and CAMSAP2 siRNA transfected hRPEs . α - tubulin is used as loading controlLeft : Images showing collagen gel deformation at 0 h , 8 h and 24 h . HRPEs transfected with negative control , CAMSAP2 and AKAP450 siRNA were seeded in 1 % collagen gel ( yellow dashed lines ) . Scale bar : 5 mm . Right : Quantification of gel area .

O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Western blot showing the protein level of active RhoA, total RhoA in negative control and CAMSAP2 siRNA transfected hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition.Left: Representative live-cell fluorescence imaging of GFP-AHPH in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of GFP-AHPH fluorescence intensity.Left: Representative immunofluorescent staining of pMLC (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of pMLC fluorescence intensity. Below: Western blot showing the protein level of pMLC in negative control and CAMSAP2 siRNA transfected hRPEs. α-tubulin is used as loading controlLeft: Images showing collagen gel deformation at 0 h, 8 h and 24 h. HRPEs transfected with negative control, CAMSAP2 and AKAP450 siRNA were seeded in 1% collagen gel (yellow dashed lines). Scale bar: 5 mm. Right: Quantification of gel area.


Figure 4Left : Representative live - cell fluorescence imaging of RFP - paxillin in control and nocodazole ( 8 μM , 30 min ) treated hRPEs . Scale bar : 20 μm . Right : Quantification of FA area obtained by FAAS . Top : Representative live - cell fluorescence imaging of RFP - paxillin in negative control and CAMSAP2 siRNA transfected hRPEs . Time - lapse fluorescence imaging zoomed from the yellow box showed the assembly and disassembly stage of a single FA . Scale bar : 20 μm . Bottom : Quantification of FA area , assembly rate and disassembly rate obtained by FAAS . Vinlin plot center lines denote the median , and dashed lines represent the 25th and 75th percentiles . Top : Schematic diagram of FA - MT contact analysis . Position of EB1 and paxillin were extracted from live - cell images , followed by automated co - localization identification . Scale bar : 20 μm . Bottom : Representative time - lapse fluorescence imaging of EGFP - EB1 and RFP - paxillin zoomed from the yellow box above showed the targeting of MT to FA . FA - MT contact ratio was calculated by the ratio of co - localization events to the total EB1 number . FA - MT contact ratio of negative control , CAMSAP2 , AKAP450 siRNA transfected and centrinone - B ( 500 nM , 7 days ) treated hRPEs . Vinlin plot center lines denote the median , and dashed lines represent the 25th and 75th percentiles . Western blot showing the protein level of ELKS in negative control and ELKS siRNA transfected hRPEs . α - tubulin was used as loading control . Bar chart shows quantification of protein levels normalized to α - tubulin in each condition . FA - MT contact ratio of negative control and ELKS siRNA transfected hRPEs . Vinlin plot center lines denote the median , and dashed lines represent the 25th and 75th percentiles . Y FMI of negative control and ELKS siRNA transfected hRPEs moving on stiffness gradient gels .

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Figure 4Left: Representative live-cell fluorescence imaging of RFP-paxillin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Quantification of FA area obtained by FAAS.Top: Representative live-cell fluorescence imaging of RFP-paxillin in negative control and CAMSAP2 siRNA transfected hRPEs. Time-lapse fluorescence imaging zoomed from the yellow box showed the assembly and disassembly stage of a single FA. Scale bar: 20 μm. Bottom: Quantification of FA area, assembly rate and disassembly rate obtained by FAAS. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.Top: Schematic diagram of FA-MT contact analysis. Position of EB1 and paxillin were extracted from live-cell images, followed by automated co-localization identification. Scale bar: 20 μm. Bottom: Representative time-lapse fluorescence imaging of EGFP-EB1 and RFP-paxillin zoomed from the yellow box above showed the targeting of MT to FA. FA-MT contact ratio was calculated by the ratio of co-localization events to the total EB1 number.   FA-MT contact ratio of negative control, CAMSAP2, AKAP450 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.   Western blot showing the protein level of ELKS in negative control and ELKS siRNA transfected hRPEs. α-tubulin was used as loading control. Bar chart shows quantification of protein levels normalized to α-tubulin in each condition.FA-MT contact ratio of negative control and ELKS siRNA transfected hRPEs. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.Y FMI of negative control and ELKS siRNA transfected hRPEs moving on stiffness gradient gels.


Figure 5Left : Representative immunofluorescent staining of GM130 ( yellow ) , F - actin ( gray ) and DAPI ( blue ) in negative control and CAMSAP2 siRNA transfected hRPEs at the edge of the wound after 8 h . Scale bar : 50 μm . Middle : Schematic diagram of Golgi - nucleus orientation towards the front ( 45 ° - 135 ° ) or the rear ( - 45 ° - - 135 ° ) . Right : Quantification of the percentage of Golgi - nucleus orientation towards the front or rear of the cell . Left : Representative immunofluorescent staining of GM130 ( yellow ) and DAPI ( blue ) in negative control and CAMSAP2 siRNA transfected hRPEs on the stiffness gradient gel . Scale bar : 20 μm . Middle : Schematic diagram of Golgi - nucleus orientation towards the stiff side ( 45 ° - 135 ° ) or the soft side ( - 45 ° - - 135 ° ) . Right : Quantification of the percentage of Golgi - nucleus orientation towards the stiff or soft side . Left : Schematic diagram of manufacturing polyacrylamide gels with islands of higher stiffness by 405 nm laser irradiation . Middle : Modulus of polyacrylamide gels with stiff islands measured by atomic force microscope . Right : Representative live - cell fluorescence imaging of cells marked with the Golgi ( green ) and nucleus ( blue ) on the soft region and the stiff island . Representative time - lapse fluorescence images showing the migration of hRPE cell to the stiff island and the reorientation of Golgi in this process . Scale bar : 20 μm . Quantification of the percentage of cells having a stiff - end biased Golgi positioning . N = 29 cells , replicates are biological .

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Figure 5Left: Representative immunofluorescent staining of GM130 (yellow), F-actin (gray) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs at the edge of the wound after 8 h. Scale bar: 50 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the front (45°-135°) or the rear (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the front or rear of the cell.Left: Representative immunofluorescent staining of GM130 (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs on the stiffness gradient gel. Scale bar: 20 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the stiff side (45°-135°) or the soft side (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the stiff or soft side.Left: Schematic diagram of manufacturing polyacrylamide gels with islands of higher stiffness by 405 nm laser irradiation. Middle: Modulus of polyacrylamide gels with stiff islands measured by atomic force microscope. Right: Representative live-cell fluorescence imaging of cells marked with the Golgi (green) and nucleus (blue) on the soft region and the stiff island.Representative time-lapse fluorescence images showing the migration of hRPE cell to the stiff island and the reorientation of Golgi in this process. Scale bar: 20 μm.   Quantification of the percentage of cells having a stiff-end biased Golgi positioning. N=29 cells, replicates are biological.


Figure 1 ( B ) B - RafE586K signals independently from Ras - GTP , but requires an intact AL . The MEK - ERK activation potential of the indicated HA - tagged B - Raf mutants was assessed by Western blotting using TCLs from transiently transfected Plat - E cells . ( C ) B - RafCAAX but not the B - RafV600E oncoprotein signals independently from Ras - GTP , but requires an intact AL . Same experimental set - up as in ( B ) . ( D ) Quantification of experiments shown in ( B ) and ( C ) . The signal elicited by the individual reference proteins ( B - Rafwt , B - RafE586K , B - RafV600E and B - RafCAAX ) was set in each analysis to 100 % . n = 3 , mean + SEM , t - test , * p < 0 . 05 , * * p < 0 .

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Figure 1(B) B-RafE586K signals independently from Ras-GTP, but requires an intact AL. The MEK-ERK activation potential of the indicated HA-tagged B-Raf mutants was assessed by Western blotting using TCLs from transiently transfected Plat-E cells.(C) B-RafCAAX but not the B-RafV600E oncoprotein signals independently from Ras-GTP, but requires an intact AL. Same experimental set-up as in (B). (D) Quantification of experiments shown in (B) and (C). The signal elicited by the individual reference proteins (B-Rafwt, B-RafE586K, B-RafV600E and B-RafCAAX) was set in each analysis to 100%. n = 3, mean + SEM, t-test, * p < 0.05, ** p < 0.01


Figure 2 ( A ) Braf - / - ; ERTmHRasG12V MEFs were reconstituted with HA - tagged hBRAF constructs or empty vector ( EV ) . Infected cells were either treated with 4 - HT ( 1µM ) to induce oncogenic Ras activation or with ethanol ( OH ) as a control . ( B ) Quantifications of pMEK ( left ) and pERK ( right ) normed to WT level . n = 3 , mean + SEM , t - test , * * p < 0 . 01 . ( C ) The AVKA mutation has no discernible influence on Ras - induced paradoxical ERK activation triggered by the D594A mutation . Experimental set - up as in ( A ) , shown are lysates from 4 - HT treated MEFs . ( D ) Quantification of pMEK and pERK levels normed to HA expression . n = 3 , mean + SEM , t - test , * * p < 0 . 01 . ( E ) Top : Braf - / - ; ERTmHRasG12V MEFs were reconstituted with the indicated B - Raf constructs . Following induction of Ras signaling , HA - B - Raf proteins were purified using anti - HA antibodies and subject to Western blotting to analyze immunoprecipitation of Raf - 1 . Bottom : Quantification of co - immunoprecipitated Raf - 1 normed to HA - B - Raf levels . n = 3 , mean + SEM , t - test , * p < 0 . 05 .

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Figure 2(A) Braf-/-;ERTmHRasG12V MEFs were reconstituted with HA-tagged hBRAF constructs or empty vector (EV). Infected cells were either treated with 4-HT (1µM) to induce oncogenic Ras activation or with ethanol (OH) as a control. (B) Quantifications of pMEK (left) and pERK (right) normed to WT level. n = 3, mean + SEM, t-test, ** p < 0.01.(C) The AVKA mutation has no discernible influence on Ras-induced paradoxical ERK activation triggered by the D594A mutation. Experimental set-up as in (A), shown are lysates from 4-HT treated MEFs. (D) Quantification of pMEK and pERK levels normed to HA expression. n = 3, mean + SEM, t-test, ** p < 0.01.(E) Top: Braf-/-;ERTmHRasG12V MEFs were reconstituted with the indicated B-Raf constructs. Following induction of Ras signaling, HA-B-Raf proteins were purified using anti-HA antibodies and subject to Western blotting to analyze immunoprecipitation of Raf-1. Bottom: Quantification of co-immunoprecipitated Raf-1 normed to HA-B-Raf levels. n = 3, mean + SEM, t-test, * p < 0.05.


Figure 3 ( B ) Genomic PCR with the primers indicated as red arrows in ( A ) showing 3 out of 9 clones being positive for homologous recombination . ( C ) Southern blot analysis of genomic DNA of parental W4 ES cells , wildtype MEFs and the ES cell clones # 286 and # 273 ( with recombined B - raffloxAVKA allele ) . Genomic DNA was digested by AseI and analyzed using the external probe indicated in ( A ) . ( D ) Genomic PCR showing Cre - mediated recombination of the BraffloxAVKA allele in vivo using the primer pair E14fwd and E15rev ( indicated as blue arrows in A ) .

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Figure 3(B) Genomic PCR with the primers indicated as red arrows in (A) showing 3 out of 9 clones being positive for homologous recombination.(C) Southern blot analysis of genomic DNA of parental W4 ES cells, wildtype MEFs and the ES cell clones #286 and #273 (with recombined B-raffloxAVKA allele). Genomic DNA was digested by AseI and analyzed using the external probe indicated in (A).(D) Genomic PCR showing Cre-mediated recombination of the BraffloxAVKA allele in vivo using the primer pair E14fwd and E15rev (indicated as blue arrows in A).


Figure 4 ( A ) Weight at weaning of BrafAVKA mice from two different founders showed no significant differences . 215 : n = WT = 24 / Heterozygous = 32 / AVKA = 19 ; 273 : n = WT = 114 / Heterozygous = 117 / AVKA = 52 , mean + SEM , t - test ( B ) Homozygous BrafAVKA mice show reduced splenic cellularity . n = 7 , mean + SEM , t - test , * p < 0 . 05 . ( C - E ) Relative number of CD8 + T cells . FACS analysis shows an increase in naïve cells ( C ) , whereas differentiated TEM ( D ) and Tregs ( E ) reveal a reduction in BrafAVKA mice compared to WT . n = 8 , mean + SEM , t - test , * p < 0 . 05 , * * p < 0 . 01 . ( F ) Primary B cells of BrafAVKA mice show reduced CD69 surface expression upon LPS and anti - IgM stimulation . n = 3 , mean + SEM , test , * * p < 0 . 01 . ( G ) Primary B cells also show reduced proliferation upon stimulation compared with WT B cells . n = 3 , mean + SEM , test , * * p < 0 .

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Figure 4(A) Weight at weaning of BrafAVKA mice from two different founders showed no significant differences. 215: n = WT = 24/ Heterozygous = 32/ AVKA = 19; 273: n = WT = 114/ Heterozygous = 117/ AVKA = 52, mean + SEM, t-test(B) Homozygous BrafAVKA mice show reduced splenic cellularity. n = 7, mean + SEM, t-test, * p < 0.05.(C-E) Relative number of CD8+ T cells. FACS analysis shows an increase in naïve cells (C), whereas differentiated TEM (D) and Tregs (E) reveal a reduction in BrafAVKA mice compared to WT. n = 8, mean + SEM, t-test, * p < 0.05, ** p < 0.01.(F) Primary B cells of BrafAVKA mice show reduced CD69 surface expression upon LPS and anti-IgM stimulation. n = 3, mean + SEM, test, ** p < 0.01.(G) Primary B cells also show reduced proliferation upon stimulation compared with WT B cells. n = 3, mean + SEM, test, ** p < 0.01


Figure 5 ( A ) Facial appearance of BrafAVKA mice . ( B ) Top : In vitro kinase ( IVK ) assay of B - Raf complexes purified from either WT or BrafAVKA knock - in brains from both founder strains revealed a reduced MEK phosphorylation potential regardless of their purification from NLB or RIPA lysates . Botttom : Quantification of IVK differentials : # 215 samples indicated in black , # 273 samples in grey . ( C ) Western blot analysis showing reduced pMEK and pERK levels in BrafAVKA brains . n = 5 , mean + SEM , t - test , * * < 0 . 01 . ( D ) Immunohistochemistry showing reduced pERK levels in Purkinje cells of homozygous BrafAVKA mice . Scale bar indicates 10x magnification .

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Figure 5(A) Facial appearance of BrafAVKA mice.(B) Top: In vitro kinase (IVK) assay of B-Raf complexes purified from either WT or BrafAVKA knock-in brains from both founder strains revealed a reduced MEK phosphorylation potential regardless of their purification from NLB or RIPA lysates. Botttom: Quantification of IVK differentials: #215 samples indicated in black, #273 samples in grey.(C) Western blot analysis showing reduced pMEK and pERK levels in BrafAVKA brains. n = 5, mean + SEM, t-test, ** < 0.01.(D) Immunohistochemistry showing reduced pERK levels in Purkinje cells of homozygous BrafAVKA mice. Scale bar indicates 10x magnification.


Figure 6 ( A ) Total brain lysates of WT and BrafAVKA mice were subject to immunoprecipitation ( IP ) using either anti - B - Raf antibodies or non - specific IgG as negative control . The co - purified proteins were visualized using the indicated antibodies and quantified . Ksr1 is significantly decreased in B - RafAVKA complexes while the other proteins are equally bound to B - RafWT or B - RafAVKA . n = 3 , mean + SEM , t - test , * p < 0 . 05 . ( B ) Anti - Myc IP of Plat - E cells transfected with Myc - tagged Raf - 1 and different HA - B - Raf constructs . Western blot analysis showed that B - RafAVKA is not impaired in its ability to interact with Raf - 1 . ( C ) Anti - Myc IP of Plat - E cells expressing different Myc - and HA - B - Raf proteins . Note that B - RafAVKA is not impaired in its ability to interact with other B - Raf proteins , while homo - dimerization of B - RafEVKD is enhanced . ( D ) Top : Exemplary Western blot analysis of EGF stimulated primary astrocytes . Mutant astrocytes show drastic reduction in basic and stimulated levels of pMEK and pERK . Bottom : Quantification of all Western blot samples . WT : n = 7 , AVKA : n = 6 , mean + SEM , t - test , * p < 0 . 05 , * * p < 0 . 01 .

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Figure 6(A) Total brain lysates of WT and BrafAVKA mice were subject to immunoprecipitation (IP) using either anti-B-Raf antibodies or non-specific IgG as negative control. The co-purified proteins were visualized using the indicated antibodies and quantified. Ksr1 is significantly decreased in B-RafAVKA complexes while the other proteins are equally bound to B-RafWT or B-RafAVKA. n = 3, mean + SEM, t-test, * p < 0.05.(B) Anti-Myc IP of Plat-E cells transfected with Myc-tagged Raf-1 and different HA-B-Raf constructs. Western blot analysis showed that B-RafAVKA is not impaired in its ability to interact with Raf-1.(C) Anti-Myc IP of Plat-E cells expressing different Myc- and HA-B-Raf proteins. Note that B-RafAVKA is not impaired in its ability to interact with other B-Raf proteins, while homo-dimerization of B-RafEVKD is enhanced.(D) Top: Exemplary Western blot analysis of EGF stimulated primary astrocytes. Mutant astrocytes show drastic reduction in basic and stimulated levels of pMEK and pERK. Bottom: Quantification of all Western blot samples. WT: n = 7, AVKA: n= 6, mean + SEM, t-test, * p <0.05, ** p < 0.01.


Figure 7 ( A ) MEFs expressing B - RafWT or B - RafAVKA were stimulated with EGF for the indicated time points ( min ) and analyzed by Western blotting . ( B ) IP of EGF stimulated MEFs expressing B - RafWT or B - RafAVKA . Sorafenib was used to increase complex build up . ( C ) Proliferation of two different MEF pools expressing either B - RafWT or B - RafAVKA . MEFs expressing B - RafAVKA show a strong reduction in growth rate . n = 3 , mean + SEM , t - test , * * p < 0 . 01 , * * * p < 0 . 001 . ( D ) Cell morphology of B - RafAVKA expressing MEFs are enlarged and present with abnormal nuclei ( E ) MEFs expressing B - RafAVKA show a significant increase in caspase - 3 activity . n = 3 , mean + SEM , t - test , * * p < 0 . 01 . ( F ) Decay of TAg expression in MEFs following dox withdrawal . ( G ) Morphology of MEFs expressing B - Rafwt or B - RafAVKA in the presence or absence of TAg .

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Figure 7(A) MEFs expressing B-RafWT or B-RafAVKA were stimulated with EGF for the indicated time points (min) and analyzed by Western blotting.(B) IP of EGF stimulated MEFs expressing B-RafWT or B-RafAVKA. Sorafenib was used to increase complex build up.(C) Proliferation of two different MEF pools expressing either B-RafWT or B-RafAVKA. MEFs expressing B-RafAVKA show a strong reduction in growth rate. n = 3, mean + SEM, t-test, ** p < 0.01, *** p < 0.001.(D) Cell morphology of B-RafAVKA expressing MEFs are enlarged and present with abnormal nuclei(E) MEFs expressing B-RafAVKA show a significant increase in caspase-3 activity. n = 3, mean + SEM, t-test, ** p < 0.01.(F) Decay of TAg expression in MEFs following dox withdrawal.(G) Morphology of MEFs expressing B-Rafwt or B-RafAVKA in the presence or absence of TAg.


Figure 8 ( A ) MEFs were then infected with retroviral vectors encoding the indicated B - Raf proteins . Giemsa staining revealed transformed and hyper - proliferated cells by dark purple colour . ( B ) Quantification of Focus formation potential of the B - Raf mutants used in ( A ) . n = 3 , mean + SEM , t - test , * * * p < 0 . 001 .

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Figure 8(A) MEFs were then infected with retroviral vectors encoding the indicated B-Raf proteins. Giemsa staining revealed transformed and hyper-proliferated cells by dark purple colour. (B) Quantification of Focus formation potential of the B-Raf mutants used in (A). n = 3, mean + SEM, t-test, *** p < 0.001.