Mice were subjected to ONC and i . v . - injected with either an anti - VCAM - 1 blocking antibody or an isotype control antibody . Eyes were collected after 2 or 3 days for Real - Time PCR analysis , respectively . ( B ) Expression ratios of Tnf , Il12a and Il1b , compared to Tgfb2 , showing that blocking VCAM - 1 results in skewing the retina towards a pro - inflammatory milieu after ONC . n = 11 - 13 per group . Data shown are representative of two independent experiments . Bar graphs throughout the figure show mean ± SE of each group . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . noninj - noninjured control ; ONC - optic nerve crush .

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

Mice were subjected to ONC and i.v.-injected with either an anti-VCAM-1 blocking antibody or an isotype control antibody. Eyes were collected after 2 or 3 days for Real-Time PCR analysis, respectively. (B) Expression ratios of Tnf, Il12a and Il1b, compared to Tgfb2, showing that blocking VCAM-1 results in skewing the retina towards a pro-inflammatory milieu after ONC. n = 11-13 per group. Data shown are representative of two independent experiments. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.


( A ) Live cell microscopy imaging of noninjured and ONC eyes , showing the presence of DiR + monocyte clusters in injured eyes only . Some of the cell clusters are co - localized with GFP , indicating monocytes that have crossed the RPE and infiltrated the retina . n = 2 - 3 per group . Data shown are representative of two independent experiments .

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

(A) Live cell microscopy imaging of noninjured and ONC eyes, showing the presence of DiR+monocyte clusters in injured eyes only. Some of the cell clusters are co-localized with GFP, indicating monocytes that have crossed the RPE and infiltrated the retina. n = 2-3 per group. Data shown are representative of two independent experiments.


( B ) Representative micrographs of eye sections collected from mice 1 day after ONC , or 29 days after the induction of EAU , stained for β - dystroglycan ( red , arrows ) and laminin ( green ) , showing a comparable structure of blood vessels in the inner retina of ONC eyes and noninjured controls , which is different from the appearance of blood vessels in EAU retina . Scale bar , 50 µm .

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

(B) Representative micrographs of eye sections collected from mice 1 day after ONC, or 29 days after the induction of EAU, stained for β-dystroglycan (red, arrows) and laminin (green), showing a comparable structure of blood vessels in the inner retina of ONC eyes and noninjured controls, which is different from the appearance of blood vessels in EAU retina. Scale bar, 50 µm.


( C - E ) [ Actbegfp / + WT ] BM chimeric mice were generated and subjected to ONC ( optic nerve crush ) . ( C ) Representative micrographs showing the abundance of GFP + cells ( green ) in the RPE ( RPE65 , red ) and retina of the ONC eye compared to noninjured contralateral eye . Retinal layers can be distinguished by Hoechst staining ( blue ; scale bar , 100 µm ) . ( D ) Higher magnification micrographs of ONC eyes , showing the association of blood - derived GFP + cells ( green , arrows ) with the RPE ( red ) ; Note processes extending from the cells to the RPE ( scale bar , 20 µm ) . ( E ) Representative micrograph of ONC eye showing GFP + cells ( green ) in proximity to the GCL , the site of dying retinal ganglion cells ( scale bar , 20 µm ) . n = 4 per group .

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

(C-E) [Actbegfp/+ WT] BM chimeric mice were generated and subjected to ONC (optic nerve crush). (C) Representative micrographs showing the abundance of GFP+ cells (green) in the RPE (RPE65, red) and retina of the ONC eye compared to noninjured contralateral eye. Retinal layers can be distinguished by Hoechst staining (blue; scale bar, 100 µm). (D) Higher magnification micrographs of ONC eyes, showing the association of blood-derived GFP+ cells (green, arrows) with the RPE (red); Note processes extending from the cells to the RPE (scale bar, 20 µm). (E) Representative micrograph of ONC eye showing GFP+ cells (green) in proximity to the GCL, the site of dying retinal ganglion cells (scale bar, 20 µm). n = 4 per group.


( A - D ) Changes in mRNA transcript levels of a series of adhesion molecules ( A ) , chemokines ( B , C ) and cytokine receptors ( D ) in injured retinas compared with contralateral noninjured retinas , assessed 1 day after GT intoxication . n = 6 per group .

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

(A-D) Changes in mRNA transcript levels of a series of adhesion molecules (A), chemokines (B, C) and cytokine receptors (D) in injured retinas compared with contralateral noninjured retinas, assessed 1 day after GT intoxication. n = 6 per group.


( E ) Representative micrographs showing the expression of ICAM - 1 and VCAM - 1 ( red ) by the RPE ( green ) 1 day after GT intoxication . Bar graphs throughout the figure show mean ± SE of each group . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . GT - glutamate ; noninj - noninjured control .

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

(E) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 1 day after GT intoxication. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. GT - glutamate; noninj - noninjured control.


( A ) Representative micrograph of an eye from day 7 after glutamate intoxication , from a mouse that was i . v . - injected with monocytes , showing monocyte localization to the SRS after this mode of application , and indicating a potential homing route for the cells ( IL - 10 , red ; CX3CR1 - GFP , green ) .

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

(A) Representative micrograph of an eye from day 7 after glutamate intoxication, from a mouse that was i.v.-injected with monocytes, showing monocyte localization to the SRS after this mode of application, and indicating a potential homing route for the cells (IL-10, red; CX3CR1-GFP, green).


( B - E ) Mice were injected i . v . t . with either monocytes or PBS 1 day after GT intoxication , and ocular sections from 7 days after the injury were analyzed . ( B , C ) Representative micrographs of noninjured and injured retinas stained for the RGC marker , Brn3a ( red ; scale bar , 100 µm ) , and quantification of RGC survival . Arrows indicate Brn3a + cells . Bar graph shows mean ± SE of each group . * , P < 0 . 05 .

O O O O O B-ORGANISM O O O O O O O O O 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 B-TISSUE O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE 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-E) Mice were injected i.v.t. with either monocytes or PBS 1 day after GT intoxication, and ocular sections from 7 days after the injury were analyzed. (B, C) Representative micrographs of noninjured and injured retinas stained for the RGC marker, Brn3a (red; scale bar, 100 µm), and quantification of RGC survival. Arrows indicate Brn3a+ cells. Bar graph shows mean ± SE of each group. *, P < 0.05.


( B - E ) Mice were injected i . v . t . with either monocytes or PBS 1 day after GT intoxication , and ocular sections from 7 days after the injury were analyzed . ( D , E ) Representative micrographs of GT - intoxicated eyes that were i . v . t . - injected with monocytes , showing the expression of an array of cytokines and neurotrophic factors ( red ) by the injected monocytes ( CX3CR1 - GFP , green ) . The injected cells were found in the vitreous and in proximity to injured RGCs ( left panels ) , as well as in the SRS , adjacent to the RPE ( right panels ) . Insets show higher magnification of representative cells ( scale bar , 100 µm ; inset scale , 20 µm ) . n = 4 - 5 per group . GCL - ganglion cell layer ; GT - glutamate ; mon - monocytes ; RGC - retinal ganglion cell ; RPE - retinal pigment epithelium ; SRS - subretinal space ; vit - vitreous .

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

(B-E) Mice were injected i.v.t. with either monocytes or PBS 1 day after GT intoxication, and ocular sections from 7 days after the injury were analyzed. (D, E) Representative micrographs of GT-intoxicated eyes that were i.v.t.-injected with monocytes, showing the expression of an array of cytokines and neurotrophic factors (red) by the injected monocytes (CX3CR1-GFP, green). The injected cells were found in the vitreous and in proximity to injured RGCs (left panels), as well as in the SRS, adjacent to the RPE (right panels). Insets show higher magnification of representative cells (scale bar, 100 µm; inset scale, 20 µm). n = 4-5 per group. GCL - ganglion cell layer; GT - glutamate; mon - monocytes; RGC - retinal ganglion cell; RPE - retinal pigment epithelium; SRS - subretinal space; vit - vitreous.


In panels ( A ) violin plots show the variability in continuous leaf 16 , ear phenotypes among the 60 individual plants used in downstream analyses . Data information : In all violin plots , the median is indicated by the white circle . The black box extends from the 25th to the 75th percentile , and black whiskers extend from each end of the box to the most extreme values within 1 . 5 times the interquartile range from the respective end . Data points beyond this range are shown as black dots . The red open circle indicates the mean of the distribution , with red whiskers extending to 1 standard deviation above and below the mean .

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

In panels (A) violin plots show the variability in continuous leaf 16, ear phenotypes among the 60 individual plants used in downstream analyses. Data information: In all violin plots, the median is indicated by the white circle. The black box extends from the 25th to the 75th percentile, and black whiskers extend from each end of the box to the most extreme values within 1.5 times the interquartile range from the respective end. Data points beyond this range are shown as black dots. The red open circle indicates the mean of the distribution, with red whiskers extending to 1 standard deviation above and below the mean.


In panels ( E ) , violin plots show the variability in plant height phenotypes among the 60 individual plants used in downstream analyses . Data information : In all violin plots , the median is indicated by the white circle . The black box extends from the 25th to the 75th percentile , and black whiskers extend from each end of the box to the most extreme values within 1 . 5 times the interquartile range from the respective end . Data points beyond this range are shown as black dots . The red open circle indicates the mean of the distribution , with red whiskers extending to 1 standard deviation above and below the mean .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In panels (E), violin plots show the variability in plant height phenotypes among the 60 individual plants used in downstream analyses. Data information: In all violin plots, the median is indicated by the white circle. The black box extends from the 25th to the 75th percentile, and black whiskers extend from each end of the box to the most extreme values within 1.5 times the interquartile range from the respective end. Data points beyond this range are shown as black dots. The red open circle indicates the mean of the distribution, with red whiskers extending to 1 standard deviation above and below the mean.


B . Genotype distribution in litters from heterozygous matings ( Fbxl4 + / - x Fbxl4 + / - ) at embryonic stage 13 . 5 ( E13 . 5 ) and of live animals at weaning . Data is presented as a percentage . Embryos n = 79 , weaned mice n = 339 . Dashed lines indicate the expected Mendelian ratios . Chi2 test vs expected Mendelian ratios ; * * * , p < 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD 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 B-EXP_ASSAY O B-ORGANISM 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. Genotype distribution in litters from heterozygous matings (Fbxl4+/- x Fbxl4+/-) at embryonic stage 13.5 (E13.5) and of live animals at weaning. Data is presented as a percentage. Embryos n=79, weaned mice n=339. Dashed lines indicate the expected Mendelian ratios. Chi2 test vs expected Mendelian ratios; ***, p<0.001.


C . Representative images of wild - type ( + / + ) , heterozygous knockout ( + / - ) and homozygous Fbxl4 knockout embryos ( - / - ) at E13 . 5 .

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

C. Representative images of wild-type (+/+), heterozygous knockout (+/-) and homozygous Fbxl4 knockout embryos (-/-) at E13.5.


D . Relative levels of mtDNA of Fbxl4 knockout ( - / - ) embryos and the corresponding wild type ( + / + ) embryos at E13 . 5 . Data represent mean ± SEM , n = 5 . Student ' s t - test ; * , p < 0 . 05 .

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

D. Relative levels of mtDNA of Fbxl4 knockout (-/-) embryos and the corresponding wild type (+/+) embryos at E13.5. Data represent mean ± SEM, n=5. Student's t-test; *, p<0.05.


E . Representative images of male Fbxl4 knockout ( - / - ) and matched wild - type ( + / + ) animals at one year of age ( left ) . A hunch - back phenotype was observed both in male and female knockout animals ( right , indicated by arrow ) .

O O O B-EXP_ASSAY 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 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

E. Representative images of male Fbxl4 knockout (-/-) and matched wild-type (+/+) animals at one year of age (left). A hunch-back phenotype was observed both in male and female knockout animals (right, indicated by arrow).


F - I . Whole body ( F ) , heart ( G ) , liver ( H ) and kidney ( I ) weights of 1 - year old animals . Data represent mean ± SEM , n ≥ 5 animals . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . , not significant ( p ≥ 0 . 05 ) .

O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O B-TISSUE O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I. Whole body (F), heart (G), liver (H) and kidney (I) weights of 1-year old animals. Data represent mean ± SEM, n ≥ 5 animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant (p ≥ 0.05).


A . Relative levels of mtDNA in tissues of 1 - year - old Fbxl4 knockout mice determined by quantitative real - time PCR ( normalized to controls ) . Data are presented as mean ± SEM , n = 6 for controls and n = 8 for knockout animals . Student ' s t - test ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

A. Relative levels of mtDNA in tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR (normalized to controls). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; **, p<0.01; ***, p<0.001.


B . Transcript levels in indicated tissues of 1 - year - old Fbxl4 knockout mice determined by quantitative real - time PCR . Relative mRNA levels in control animals were set as 1 ( dashed line ) . Data are presented as mean ± SEM , n = 6 for controls and n = 8 for knockout animals . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O O O O 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. Transcript levels in indicated tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR. Relative mRNA levels in control animals were set as 1 (dashed line). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001.


C . Western blot analysis of protein steady - state levels in liver protein extracts from wild type ( Fbxl4 + / + ) and Fbxl4 knock - out ( Fbxl4 - / - ) animals . D . Quantification of the western blot in ( C ) . Signal normalized to the average of controls and presented as mean ± SEM , n = 4 for both controls and knockout animals . Student ' s t - test ; * * , p < 0 . 01 ; * * * , p < 0 , 001 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O 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 O O O O O O O O O O O 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 analysis of protein steady-state levels in liver protein extracts from wild type (Fbxl4+/+) and Fbxl4 knock-out (Fbxl4-/-) animals. D. Quantification of the western blot in (C). Signal normalized to the average of controls and presented as mean ± SEM, n=4 for both controls and knockout animals. Student's t-test; **, p<0.01; ***, p<0,001.


E . Quantitative TMT - based proteomic analysis of wild - type and Fbxl4 knock - out liver tissue protein extracts . Proteins from three control and three Fbxl4 knock - out liver protein extracts were TMT - labelled , quantified and analyzed separately as described in Materials and Methods ; the data from control and knock - out animals were averaged after the analysis and presented as - log10 of the adjusted p - value vs log2 - fold change ( logFC ) . Mitochondrial proteins were selected according to mouse MitoCarta 2 . 0 ( Calvo et al , 2016 ) and highlighted in red ; lysosomal proteins were selected according to hLGDB database ( Brozzi et al , 2013 ) and highlighted in yellow .

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

E. Quantitative TMT-based proteomic analysis of wild-type and Fbxl4 knock-out liver tissue protein extracts. Proteins from three control and three Fbxl4 knock-out liver protein extracts were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and knock-out animals were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to mouse MitoCarta 2.0(Calvo et al, 2016) and highlighted in red; lysosomal proteins were selected according to hLGDB database(Brozzi et al, 2013) and highlighted in yellow.


F . Enrichment analysis of lysosomal and mitochondrial proteins according to the logFC median of each group of proteins . Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing the ± 1 . 5x inter - quartile range . Proteins included in each group : total = 1523 , mitochondrial = 324 , lysosomal = 50 .

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

F. Enrichment analysis of lysosomal and mitochondrial proteins according to the logFC median of each group of proteins. Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing the ±1.5x inter-quartile range. Proteins included in each group: total=1523, mitochondrial=324, lysosomal=50.


B . Relative mtDNA content in fibroblasts from patients and controls as determined by qRT - PCR using an ND6 probe and normalized to nuclear genomic DNA content using an 18S probe . Individual values are presented for each sample , as well as their mean ± SEM values .

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

B. Relative mtDNA content in fibroblasts from patients and controls as determined by qRT-PCR using an ND6 probe and normalized to nuclear genomic DNA content using an 18S probe. Individual values are presented for each sample, as well as their mean ± SEM values.


C . Steady - state protein levels in three control and three FBXL4 patient - derived fibroblasts analyzed by western blotting .

C. Steady-state protein levels in three control and three FBXL4 patient-derived fibroblasts analyzed by western blotting.


D . Quantification of the western blot in ( C ) . Signals from the control and FBXL4 - mutated cells were considered as biological replicates , normalized to the average of controls and displayed as mean ± SEM ( n = 3 for each ) . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

O 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 O O O O O O O O O O O O O O O O O O O O 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 western blot in (C). Signals from the control and FBXL4-mutated cells were considered as biological replicates, normalized to the average of controls and displayed as mean ± SEM (n=3 for each). Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001.


E . Quantification of nuclear and mitochondrial transcript levels in control and patient fibroblasts . The results from control and patient cells were averaged , normalized to controls and displayed as mean ± SEM ( n = 3 biological replicates for each condition ) .

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

E. Quantification of nuclear and mitochondrial transcript levels in control and patient fibroblasts. The results from control and patient cells were averaged, normalized to controls and displayed as mean ± SEM (n=3 biological replicates for each condition).


F . Quantitative TMT - based proteomic analysis of control and patient fibroblasts . Proteins from three control and three patient fibroblast cell lines were TMT - labelled , quantified and analyzed separately as described in Materials and Methods ; the data from control and patients were averaged after the analysis and presented as - log10 of the adjusted p - value vs log2 - fold change ( logFC ) . Mitochondrial proteins were selected according to human MitoCarta 2 . 0 ( Calvo et al , 2016 ) and highlighted in blue ; lysosomal proteins were selected according to hLGDB database ( Brozzi et al , 2013 ) and highlighted in yellow .

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F. Quantitative TMT-based proteomic analysis of control and patient fibroblasts. Proteins from three control and three patient fibroblast cell lines were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and patients were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to human MitoCarta 2.0 (Calvo et al, 2016) and highlighted in blue; lysosomal proteins were selected according to hLGDB database (Brozzi et al, 2013) and highlighted in yellow.


G . Log2FC correlation analysis of Mouse Liver proteomic dataset and Fibroblast Proteomic dataset . Proteins with a log2FC > 0 . 5 are highlighted in black and lysosomal proteins are highlighted in yellow .

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G. Log2FC correlation analysis of Mouse Liver proteomic dataset and Fibroblast Proteomic dataset. Proteins with a log2FC > 0.5 are highlighted in black and lysosomal proteins are highlighted in yellow.


B . Steady - state protein levels in wild - type ( WT ) and FBXL4 knockout ( KO ) RKO cells .

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B. Steady-state protein levels in wild-type (WT) and FBXL4 knockout (KO) RKO cells.


C . ( Upper panel ) Pulse - chase labeling of mitochondrial translation products in wild type ( WT ) and FBXL4 knockout ( KO ) cells . The cells were incubated for 1 hour with a 35S methionine - cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation ( pulse ) . After removal of the medium , the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours ( chase ) . A representative experiment of 4 biological replicates is shown . A Coomassie - stained part of the gel is shown as loading control . ( Lower panel ) Quantification of the MTCO1 - ND4 signal presented as mean ± SEM ; n = 4 biological replicates ; Student ' s t - test ; * , p < 0 . 05 .

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C. (Upper panel) Pulse-chase labeling of mitochondrial translation products in wild type (WT) and FBXL4 knockout (KO) cells. The cells were incubated for 1 hour with a 35S methionine-cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation (pulse). After removal of the medium, the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours (chase). A representative experiment of 4 biological replicates is shown. A Coomassie-stained part of the gel is shown as loading control. (Lower panel) Quantification of the MTCO1-ND4 signal presented as mean ± SEM; n=4 biological replicates; Student's t-test; *, p<0.05.


D . ( Upper panel ) Mitochondrial translation products were labeled in wild type ( WT ) and FBXL4 knock - out ( KO ) RKO cells for 60 minutes as in C , followed by 24 h chase in the presence of 5 μM epoxomicin ( epoxo ) or 20 mM ammonium chloride ( NH4Cl ) . A representative experiment of n ≥ 3 biological replicates is shown . A fragment of the Coomassie - stained gel is shown as loading control . ( Lower panel ) Quantification of the ND4 - MTCO1 signal . n ≥ 3 biological replicates , data are presented as mean ± SEM . Student ' s t - test ; * , p < 0 . 05 .

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

D. (Upper panel) Mitochondrial translation products were labeled in wild type (WT) and FBXL4 knock-out (KO) RKO cells for 60 minutes as in C, followed by 24 h chase in the presence of 5 μM epoxomicin (epoxo) or 20 mM ammonium chloride (NH4Cl). A representative experiment of n≥3 biological replicates is shown. A fragment of the Coomassie-stained gel is shown as loading control. (Lower panel) Quantification of the ND4-MTCO1 signal. n≥3 biological replicates, data are presented as mean ± SEM. Student's t-test; *, p<0.05.


E - F . Steady - state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with

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E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with NH4Cl


E - F . Steady - state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin ( Epoxo , F ) .

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E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin (Epoxo, F).


A . Confocal images from the mito - QC - expressing fibroblast lines . Scale bar 25 μm . B . Quantification of the mitolysosomes / cell area ( μm2 ) using the mito - QC counter plugin for ImageJ ( Montava - Garriga et al , 2020 ) . At least 3 technical replicates for each line and n > 15 cells analyzed for each technical replicate . Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing 5 - 95 percentile interval . Non - parametric Mann - Whitney test ; * * * , p < 0 . 001 .

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A. Confocal images from the mito-QC-expressing fibroblast lines. Scale bar 25 μm. B. Quantification of the mitolysosomes/cell area (μm2) using the mito-QC counter plugin for ImageJ (Montava-Garriga et al, 2020). At least 3 technical replicates for each line and n>15 cells analyzed for each technical replicate. Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing 5-95 percentile interval. Non-parametric Mann-Whitney test; ***, p<0.001.


( B ) Representative confocal microscopy sections of m6A - Tracer signal in HAP - 1 cells following pA - DamID with indicated antibodies . pA - Dam only : primary antibody was omitted , the dotted yellow line indicates DAPI segmentation . Free Dam : permeabilized cells were treated with freely diffusing pure Dam protein and SAM for 30 min . Scale bar corresponds to 2 µm . ( C ) Quantification of peripheral enrichment of antibody staining and m6A - Tracer signals after pA - DamID with indicated antibodies . The nuclear rim and interior were segmented using DAPI signal and the mean m6A or antibody signal was determined and tranformed to a log2 - ratio . The nuclear rim mask extends slightly beyond the NL , resulting in underestimation of the real enrichment .

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(B) Representative confocal microscopy sections of m6A-Tracer signal in HAP-1 cells following pA-DamID with indicated antibodies. pA-Dam only: primary antibody was omitted, the dotted yellow line indicates DAPI segmentation. Free Dam: permeabilized cells were treated with freely diffusing pure Dam protein and SAM for 30 min. Scale bar corresponds to 2 µm. (C) Quantification of peripheral enrichment of antibody staining and m6A-Tracer signals after pA-DamID with indicated antibodies. The nuclear rim and interior were segmented using DAPI signal and the mean m6A or antibody signal was determined and tranformed to a log2-ratio. The nuclear rim mask extends slightly beyond the NL, resulting in underestimation of the real enrichment.


( D ) HT1080 cells expressing inducible Dam - Lamin B1 were treated with Shield1 to induce m6A methylation . Cells were either fixed immediately or processed according to the pA - DamID protocol ( as negative controls ) and then fixed on poly - L - lysine coated cover slips . Cells were imaged by confocal microscopy for the NL ( Lamin B2 antibody ) and methylation ( m6A - Tracer ) for both conditions . Laser settings were changed between images to optimize image quality . The mask for defining the nuclear rim was obtained by segmentation of the DAPI image . The scale bar corresponds to 2 µm .

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

(D) HT1080 cells expressing inducible Dam-Lamin B1 were treated with Shield1 to induce m6A methylation. Cells were either fixed immediately or processed according to the pA-DamID protocol (as negative controls) and then fixed on poly-L-lysine coated cover slips. Cells were imaged by confocal microscopy for the NL (Lamin B2 antibody) and methylation (m6A-Tracer) for both conditions. Laser settings were changed between images to optimize image quality. The mask for defining the nuclear rim was obtained by segmentation of the DAPI image.The scale bar corresponds to 2 µm.


( E ) For every cell , the 50 % decay distance from the nuclear periphery was determined for Lamin B2 and m6A - Tracer by fitting exponential decay functions from the nuclear rim as defined by segmentation of the DAPI image The difference in 50 % decay distance between Lamin B2 and m6A - Tracer was used as a measure of the thickness of the m6A - Tracer layer .

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(E) For every cell, the 50% decay distance from the nuclear periphery was determined for Lamin B2 and m6A-Tracer by fitting exponential decay functions from the nuclear rim as defined by segmentation of the DAPI image The difference in 50% decay distance between Lamin B2 and m6A-Tracer was used as a measure of the thickness of the m6A-Tracer layer.


( F ) Distribution of the m6A - Tracer layer thickness for HT1080 cells expressing Dam - Lamin B1 , visualized before and after the pA - DamID protocol . For comparison , a similar analysis was performed with HT1080 cells not expressing Dam - LaminB1 , subjected to Lamin B2 pA - DamID .

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(F) Distribution of the m6A-Tracer layer thickness for HT1080 cells expressing Dam-Lamin B1, visualized before and after the pA-DamID protocol. For comparison, a similar analysis was performed with HT1080 cells not expressing Dam-LaminB1, subjected to Lamin B2 pA-DamID.


( A ) Example of raw pA - DamID data tracks from 2 million HAP - 1 cells for Lamin B2 , H3K27me3 and the Dam control from a single experiment . Sequenced reads are counted in 20kb bins and normalized for library size . ( B ) Same pA - DamID tracks of Lamin B2 and H3K27me3 as in ( A ) but after normalization to the Dam - only control ( to correct for accessibility and amplification biases ) and log2 - transformation .

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(A) Example of raw pA-DamID data tracks from 2 million HAP-1 cells for Lamin B2, H3K27me3 and the Dam control from a single experiment. Sequenced reads are counted in 20kb bins and normalized for library size. (B) Same pA-DamID tracks of Lamin B2 and H3K27me3 as in (A) but after normalization to the Dam-only control (to correct for accessibility and amplification biases) and log2-transformation.


( C ) Correlation between the median peripheral enrichment of m6A - Tracer determined by confocal microscopy and enrichment of sequencing reads within LADs in HAP - 1 cells . The LAD definition is based on conventional Lamin B1 DamID data . Every point represents a single pA - DamID experiment for which both microscopy and sequencing data were generated , colored by the antibody used . The blue line represents a linear model with a standard error confidence interval in grey . The red dashed line represents the diagonal . The Pearson correlation coefficient was converted to a t - statistic and a one - sided t - test with n - 2 degrees of freedom was used to determine statistical significance .

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(C) Correlation between the median peripheral enrichment of m6A-Tracer determined by confocal microscopy and enrichment of sequencing reads within LADs in HAP-1 cells. The LAD definition is based on conventional Lamin B1 DamID data. Every point represents a single pA-DamID experiment for which both microscopy and sequencing data were generated, colored by the antibody used. The blue line represents a linear model with a standard error confidence interval in grey. The red dashed line represents the diagonal. The Pearson correlation coefficient was converted to a t-statistic and a one-sided t-test with n-2 degrees of freedom was used to determine statistical significance.


( D ) Comparisons of pA - DamID genome - wide data ( bin size 20 kb ) for different lamin antibodies in hTERT - RPE cells : Lamin B1 vs . Lamin B2 ( left panel ) and Lamin B2 vs . Lamin A / C ( right panel ) . n denotes the number of independent biological experiments that were averaged for each data track . The blue line represents a linear model . The red dashed line represents the diagonal .

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(D) Comparisons of pA-DamID genome-wide data (bin size 20 kb) for different lamin antibodies in hTERT-RPE cells: Lamin B1 vs. Lamin B2 (left panel) and Lamin B2 vs. Lamin A/C (right panel). n denotes the number of independent biological experiments that were averaged for each data track. The blue line represents a linear model. The red dashed line represents the diagonal.


( A ) Comparison of pA - DamID and DamID data for for various lamins across a representative genomic locus in HAP - 1 and hTERT - RPE cells . Arrow indicates a region with dissimilar pA - DamID and DamID scores .

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(A) Comparison of pA-DamID and DamID data for for various lamins across a representative genomic locus in HAP-1 and hTERT-RPE cells. Arrow indicates a region with dissimilar pA-DamID and DamID scores.


( B ) Genome - wide correlation of Lamin B1 pA - DamID and Lamin B1 DamID data . The blue line represents a linear model . The red dashed line represents the diagonal .

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(B) Genome-wide correlation of Lamin B1 pA-DamID and Lamin B1 DamID data. The blue line represents a linear model. The red dashed line represents the diagonal.


( D ) Multidimensional scaling ( MDS ) plot showing all individual replicates of DamID and pA - DamID experiments for various lamins and cell types .

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(D) Multidimensional scaling (MDS) plot showing all individual replicates of DamID and pA-DamID experiments for various lamins and cell types.


( B ) NL interactions in hTERT - RPE cells at indicated times after mitosis for chromosome 2 , as determined by Lamin B2 pA - DamID . Log2 - ratios were convered to z - scores to account for differences in dynamic range between replicates Average of two independent biological replicates . LADs are colored by their differential class ( down : red , stable : grey , up : green , The centromere is shown as grey box .

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(B) NL interactions in hTERT-RPE cells at indicated times after mitosis for chromosome 2, as determined by Lamin B2 pA-DamID. Log2-ratios were convered to z-scores to account for differences in dynamic range between replicates Average of two independent biological replicates. LADs are colored by their differential class (down:red, stable:grey, up:green, The centromere is shown as grey box.


( C ) pA - DamID signal as a function of time after mitosis , for stable ( grey , n = 717 ) and dynamic LADs ( red and green : decreasing ( n = 234 ) and increasing ( n = 225 ) NL interactions , respectively ) . Lines represent mean signals of the three LAD classes and the shaded area one standard deviation on both sides . ( D ) Rate of changes in NL interactions ( average pA - DamID z - score per hour ) as function of time after mitosis for stable and dynamics LADs Boxplots : horizontal lines represent 25th , 50th and 75th percentiles ; whiskers extend to the smallest values no further than 1 . 5 times distance between 25th and 75th percentiles .

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(C) pA-DamID signal as a function of time after mitosis, for stable (grey, n=717) and dynamic LADs (red and green: decreasing (n=234) and increasing (n=225) NL interactions, respectively). Lines represent mean signals of the three LAD classes and the shaded area one standard deviation on both sides. (D) Rate of changes in NL interactions (average pA-DamID z-score per hour) as function of time after mitosis for stable and dynamics LADs Boxplots: horizontal lines represent 25th, 50th and 75th percentiles; whiskers extend to the smallest values no further than 1.5 times distance between 25th and 75th percentiles.


( E - G ) LAD size distribution in basepairs ( E ) , density of active genes ( F ) and Repli - seq score distributions ( G ) for stable and dynamic LADs Statistical significance was determined using a two ‐ way Wilcoxin test with Benjamini - Hochberg p - value correction to account for multiple testing .

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(E-G) LAD size distribution in basepairs (E), density of active genes (F) and Repli-seq score distributions (G) for stable and dynamic LADs Statistical significance was determined using a two‐way Wilcoxin test with Benjamini-Hochberg p-value correction to account for multiple testing.


( H ) Difference in pA - DamID z - scores between 1h and 21h time points , plotted against the distance to telomeres ( leftpanel ) or centromeres ( rightpanel ) . Each dot represents a single LAD . Chromosomes are colored and ordered by size . Red line is a fitted loess curve . The Spearman correlation coefficient ( ρ ) was used to test for a significant monotonic , non - linear association .

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(H) Difference in pA-DamID z-scores between 1h and 21h time points, plotted against the distance to telomeres (leftpanel) or centromeres (rightpanel). Each dot represents a single LAD. Chromosomes are colored and ordered by size. Red line is a fitted loess curve. The Spearman correlation coefficient (ρ) was used to test for a significant monotonic, non-linear association.


Difference in pA - DamID z - scores plotted against the distance to telomeres ( leftpanel ) or centromeres ( rightpanel ) . Each dot represents a single LAD . Chromosomes are colored and ordered by size . Red line is a fitted loess curve . The Spearman correlation coefficient ( ρ ) was used to test for a significant monotonic , non - linear association . pA - DamID z - scores for individual time points .

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Difference in pA-DamID z-scores plotted against the distance to telomeres (leftpanel) or centromeres (rightpanel). Each dot represents a single LAD. Chromosomes are colored and ordered by size. Red line is a fitted loess curve. The Spearman correlation coefficient (ρ) was used to test for a significant monotonic, non-linear association. pA-DamID z-scores for individual time points.


( A ) Lamin B2 pA - DamID z - scores of a representative locus in HAP - 1 cells sorted for G1 , mid - S and G2 phases . Arrows point to regions that lose NL interaction from G1 to G2 . The red star indicates the end of the chromosome . Average of three biological replicates .

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(A) Lamin B2 pA-DamID z-scores of a representative locus in HAP-1 cells sorted for G1, mid-S and G2 phases. Arrows point to regions that lose NL interaction from G1 to G2. The red star indicates the end of the chromosome. Average of three biological replicates.


difference in pA - DamID z - scores between G2 and G1 sorted pools as function of distance to either telomere or centromere for HAP - 1 ( n = 3 ) , HCT116 ( n = 2 ) and K562 cells ( n = 2 ) .

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difference in pA-DamID z-scores between G2 and G1 sorted pools as function of distance to either telomere or centromere for HAP-1 (n=3), HCT116 (n=2) and K562 cells (n=2).


( C - E ) LAD size distribution in basepairs ( C ) , density of active genes ( D ) and Repli - seq scores ( E ) for stable and dynamic LADs . Repli - seq data are not available for HAP - 1 cells .

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

(C-E) LAD size distribution in basepairs (C), density of active genes (D) and Repli-seq scores (E) for stable and dynamic LADs. Repli-seq data are not available for HAP-1 cells.


( F ) Overlapping fractions of stable and dynamic LADs and iLADs with Hi - C subcompartments in HAP - 1 and K562 cells , as imputed by SNIPER A permutation test with 1000 permutations ( R - package regioneR was used to test for a significant enrichment of B1 subcompartments in decreasing LADs , resulting in P - values of 1e - 3 and 1e - 3 for HAP - 1 and K562 , respectively .

O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O

(F) Overlapping fractions of stable and dynamic LADs and iLADs with Hi-C subcompartments in HAP-1 and K562 cells, as imputed by SNIPER A permutation test with 1000 permutations (R-package regioneR was used to test for a significant enrichment of B1 subcompartments in decreasing LADs, resulting in P-values of 1e-3 and 1e-3 for HAP-1 and K562, respectively.


( 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 .

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 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

(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 the cell 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 O B-EXP_ASSAY I-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 . 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 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 O B-CELL_LINE O B-CELL_LINE O 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 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD 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 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 the pA - DamID signal in G1 ( left - hand panels ) or G2 cells ( right - hand panels )

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(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 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 .

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 O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 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 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 .

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 O B-CELL_TYPE O O O O B-GENEPROD O 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 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 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 .

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

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 O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O 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 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 ) 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 .

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 O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE O O 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 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 .

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 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 O B-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 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 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 .

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 O B-SMALL_MOLECULE O 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 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 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 ,

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-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 O B-TISSUE I-TISSUE O 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 O O B-GENEPROD O O O O O O B-GENEPROD O 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 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 ) .

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

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

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O 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 O B-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 O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O 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 ) 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 ,

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

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

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 the indicated timepoints . 80 % of the applied C10M mass was excreted after 90

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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 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 .

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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 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 ) .

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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 .

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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 .

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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 .

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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 .

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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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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 , 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 .

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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 ) . The depicted C10M control without CRP shows no clinical signs of rejection after five days ( right ) .

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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 .

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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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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 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 .

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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 ) 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 .

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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 .

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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 .

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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 ) 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 .

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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 . 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 .

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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.