Figure 1B . 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 . 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 ) . 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 ) .

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Figure 1B. 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.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).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).


Figure 2A . 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 . 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 . 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 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 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 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 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 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

Figure 2A. 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.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.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.


Figure 3B . 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 . 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 ) . 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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Figure 3B. 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.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).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.


Figure 4B . 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 . 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 NH4ClE - F . Steady - state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin ( Epoxo , F ) .

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Figure 4B. 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.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 NH4ClE-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin (Epoxo, F).


Figure 5A . 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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Figure 5A. 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.


Figure 1 ( 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 . ( 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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Figure 1(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.(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.


Figure 2 ( 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 . ( 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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Figure 2(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.(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.


Figure 3 ( 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 . ( 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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Figure 3(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.(D) Multidimensional scaling (MDS) plot showing all individual replicates of DamID and pA-DamID experiments for various lamins and cell types.


Figure 4 ( 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 . ( 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 . 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 .

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 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 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 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 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

Figure 4(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.(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.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.


Figure 5 ( 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 ) . ( 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 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 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 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 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

Figure 5(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).(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.


Figure 6 ( 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 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 ) . ( D ) Scatterplots of changes in pA - DamID signal versus the pA - DamID signal in G1 ( left - hand panels ) or G2 cells ( right - hand panels )

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 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 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 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

Figure 6(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 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).(D) Scatterplots of changes in pA-DamID signal versus the pA-DamID signal in G1 (left-hand panels) or G2 cells (right-hand panels)


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 ) .

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 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 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

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 .

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 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 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 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 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 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 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 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

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 .

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 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 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 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

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 1B . 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 . D . Quantification of mRNA levels in hippocampi of heterozygous and homozygous hTauAT 16 month old mice ( n = 4 per group ) . E . Distribution of hTauAT visualized by the human Tau specific antibody HT7 in the hippocampal CA3 - region of hetero - and homozygous hTauATmice at the age of 14 month . Notice the mis - sorted hTauAT in cell bodies and dendrites ( arrowheads ) of pyramidal neurons of the CA3 region and immunreactivity of the axons ( mossy fibers , asterisks ) . By contrast the control shows no immunoreactivity . F . Elevated hTauAT - expression causes Tau - pathology inside area CA3 of the hippocampus . Note the increase in Tau phosphorylation probed with the antibody against pT217 , a site upstream of the repeat domain . Arrowheads point to areas with phosphorylated and mislocalized Tau in cell somata of pyramidal neurons in area CA3 . G . Tauopathy detected by antibody AT180 specific for phosphorylation sites pT231 + pSer235 ( arrowheads denote somato - dendritic mislocalized tau ) . H . Antibody PHF - 1 ( phosphorylation sites pSer396 + pSer404 ) illustrates pathological phosphorylation of Tau due to hTauAT expression especially in stratum lucidum ( asterisks ) of area CA3 and pyramidal neuroncell bodies ( arrows ) . I . Tau phosphorylation detected by antibody AT8 ( pS202 + pT205 ) in stratum pyramidale of area CA3 ( arrowhead ) . Note that immunoreactivity increases with expression level .

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 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 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 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 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 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 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 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

Figure 1B. 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.D. Quantification of mRNA levels in hippocampi of heterozygous and homozygous hTauAT 16 month old mice (n = 4 per group).E. Distribution of hTauAT visualized by the human Tau specific antibody HT7 in the hippocampal CA3-region of hetero- and homozygous hTauATmice at the age of 14 month. Notice the mis-sorted hTauAT in cell bodies and dendrites (arrowheads) of pyramidal neurons of the CA3 region and immunreactivity of the axons (mossy fibers, asterisks). By contrast the control shows no immunoreactivity.F. Elevated hTauAT -expression causes Tau-pathology inside area CA3 of the hippocampus. Note the increase in Tau phosphorylation probed with the antibody against pT217, a site upstream of the repeat domain. Arrowheads point to areas with phosphorylated and mislocalized Tau in cell somata of pyramidal neurons in area CA3.G. Tauopathy detected by antibody AT180 specific for phosphorylation sites pT231+pSer235 (arrowheads denote somato-dendritic mislocalized tau).H. Antibody PHF-1 (phosphorylation sites pSer396+pSer404) illustrates pathological phosphorylation of Tau due to hTauAT expression especially in stratum lucidum (asterisks) of area CA3 and pyramidal neuroncell bodies (arrows).I. Tau phosphorylation detected by antibody AT8 (pS202+pT205) in stratum pyramidale of area CA3 (arrowhead). Note that immunoreactivity increases with expression level.


Figure 2A . Conformational change of Tau detected by ALZ - 50 antibody immunoreactivity in stratum lucidum ( asterisks ) of area CA3 and somata in stratum pyramidale ( arrowheads ) . B . Tau aggregation confirmed by Gallyas silver staining of NFTs bearing neurons in hetero - and homozygous hTauAT mice at 14 month of age compared to non - reactive control littermate mice . In homozygous hTauAT mice the extent of neurofibrillary tangles ( NFTs ) visualized by Gallyas silver staining is enhanced compared to heterozygous hTauAT mice ( white arrowheads ) . The control shows no silver - reactive Tau aggregates . C . Western blot analysis using the pan - Tau antibody K9JA indicates sarcosyl - insoluble Tau species of human hTauAT ( upper band ) and mouse Tau ( mTau , lower band ) . Note the enhanced Tau aggregation in homozygous hTauAT mice compared to heterozygous hTauAT mice and control mice . D . Quantification of ( C ) . The ratio of hTauAT / mTau indicates a stronger aggregation in the homozygous hTauAT mice ( ~ 1 . 4 ) compared to heterozygous hTauAT mice ( ~ 0 . 7 ( n = 4 animals , error bars represent SEM ) . E . Electron micrograph of CA3 region of the hippocampus illustrating typical synapse with a perforated postsynaptic density ( asterisks ) in a control animal at 12 months . ( ds ) dendriticspine , ( pt ) presynaptic terminal and ( m ) mitochondrium . F . Example of a degenerating synaptic bouton ( arrow ) containing swollen synaptic vesicles in proximity to a large terminal with a perforated postsynaptic density ( asterisks ) in a transgenic animal at 13 months . G . Electron dense neuronal cytoplasm with dark nucleoplasm and various vacuoles ( arrow ) detected in CA3pyramidal neurons ( arrow ) at 13 months in transgenic animals in proximity to normally appearing neurons ( asterisks ) , degenerating apical dendrite ( d ) . H . Neuropil of a transgenic animal ( 13 months ) with a degenerating neuritic profile ( arrow ) . I . Degenerating dendrite ( arrow ) in a transgenic animal with darkened cytoplasm and abnormally distributed mitochondria ( arrow ) . J . Degenerating dendrite containing electron - dense whorling membrane fragments ( arrow ) in proximity to normally appearing synapses ( synapse with a perforated postsynaptic density , asterisks ) .

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

Figure 2A. Conformational change of Tau detected by ALZ-50 antibody immunoreactivity in stratum lucidum (asterisks) of area CA3 and somata in stratum pyramidale (arrowheads).B. Tau aggregation confirmed by Gallyas silver staining of NFTs bearing neurons in hetero- and homozygous hTauAT mice at 14 month of age compared to non-reactive control littermate mice. In homozygous hTauAT mice the extent of neurofibrillary tangles (NFTs) visualized by Gallyas silver staining is enhanced compared to heterozygous hTauAT mice (white arrowheads). The control shows no silver-reactive Tau aggregates.C. Western blot analysis using the pan-Tau antibody K9JA indicates sarcosyl-insoluble Tau species of human hTauAT (upper band) and mouse Tau (mTau, lower band). Note the enhanced Tau aggregation in homozygous hTauAT mice compared to heterozygous hTauAT mice and control mice.D. Quantification of (C). The ratio of hTauAT /mTau indicates a stronger aggregation in the homozygous hTauAT mice (~1.4) compared to heterozygous hTauAT mice (~0.7 (n=4 animals, error bars represent SEM).E. Electron micrograph of CA3 region of the hippocampus illustrating typical synapse with a perforated postsynaptic density (asterisks) in a control animal at 12 months. (ds) dendriticspine, (pt) presynaptic terminal and (m) mitochondrium.F. Example of a degenerating synaptic bouton (arrow) containing swollen synaptic vesicles in proximity to a large terminal with a perforated postsynaptic density (asterisks) in a transgenic animal at 13 months.G. Electron dense neuronal cytoplasm with dark nucleoplasm and various vacuoles (arrow) detected in CA3pyramidal neurons (arrow) at 13 months in transgenic animals in proximity to normally appearing neurons (asterisks), degenerating apical dendrite (d).H. Neuropil of a transgenic animal (13 months) with a degenerating neuritic profile (arrow).I. Degenerating dendrite (arrow) in a transgenic animal with darkened cytoplasm and abnormally distributed mitochondria (arrow).J. Degenerating dendrite containing electron-dense whorling membrane fragments (arrow) in proximity to normally appearing synapses (synapse with a perforated postsynaptic density, asterisks).


Figure 3A . Western blot analysis with pan - Tau K9JA antibody to estimate expression levels of hTauAT and endogenous Tau ( mTau ) . Slice homogenates from non - transgenic littermate ( lane 1 , Ctrl ) , heterozygouse hTauAT ( lane 2 , ( + / - ) ) or homozygouse hTauAT slices ( lane 3 , ( + / + ) ) were analyzed after three weeks in culture . Actin served as a loading control . B . Quantification of hTauAT expression levels ( n = 3 - 6 slice homogenates ) . The ratio between hTauAT and endogenous mouse Tau is shown . C . Immunohistochemistry with antibodies against human Tau ( TauY9 antibodies ; middle panel ) and dendritic marker MAP2 ( left panel ) in slice cultures at DIV 10 . Stratum radiatum of area CA3 is shown . Although MAP2 staining is apparent , no signal is detected by the TauY9 antibody in control slices . In contrast , in hTauAT slices human Tau is detected in both dendrites and axons ( see asterisk in merged image ) . D . Immunohistochemistry against phosphorylated Tau at the PHF1 - epitope in area CA3 of a control ( upper lane ) or hTauAT slice ( bottom lane ) . PHF1 staining is slightly visible in some axons in control slices . In contrast , PHF1 - phosphorylated Tau is apparent in somata , dendrites and axons of neurons in hTauAT slice cultures . E . Higher magnification image of apical dendrites in area CA3 in a hTauAT slice after immunohistochemistry against MAP2 ( red ) and Tau ( K9JA antibody , green ) . Tau staining is seen in dendritic spines ( asterisks ) in contrast to MAP2 , which is restricted to the dendritic shaft .

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 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 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 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

Figure 3A. Western blot analysis with pan-Tau K9JA antibody to estimate expression levels of hTauAT and endogenous Tau (mTau). Slice homogenates from non-transgenic littermate (lane 1, Ctrl), heterozygouse hTauAT (lane 2, (+/-)) or homozygouse hTauAT slices (lane 3, (+/+)) were analyzed after three weeks in culture. Actin served as a loading control.B. Quantification of hTauAT expression levels (n=3-6 slice homogenates). The ratio between hTauAT and endogenous mouse Tau is shown.C. Immunohistochemistry with antibodies against human Tau (TauY9 antibodies; middle panel) and dendritic marker MAP2 (left panel) in slice cultures at DIV 10. Stratum radiatum of area CA3 is shown. Although MAP2 staining is apparent, no signal is detected by the TauY9 antibody in control slices. In contrast, in hTauAT slices human Tau is detected in both dendrites and axons (see asterisk in merged image).D. Immunohistochemistry against phosphorylated Tau at the PHF1-epitope in area CA3 of a control (upper lane) or hTauAT slice (bottom lane). PHF1 staining is slightly visible in some axons in control slices. In contrast, PHF1-phosphorylated Tau is apparent in somata, dendrites and axons of neurons in hTauAT slice cultures.E. Higher magnification image of apical dendrites in area CA3 in a hTauAT slice after immunohistochemistry against MAP2 (red) and Tau (K9JA antibody, green). Tau staining is seen in dendritic spines (asterisks) in contrast to MAP2, which is restricted to the dendritic shaft.


Figure 4A . Slices ( DIV15 ) from control mice were loaded with the Ca + + sensitive dye Fura - 2AM . Ratiometric images are presented at baseline and after application of high potassium . Note the increase in intracellular Ca + + ( [ Ca + + ] i ) after KCl application . B . Slices ( DIV15 ) from hTauAT mice were loaded with the Ca + + sensitive dye Fura - 2AM . Ratiometric images at baseline and after application of high potassium in area CA3 are depicted . Note the increase in [ Ca + + ] i ( false color legend ) under both conditions in hTauAT slices compared to controls . C . Electrophysiological example traces ( control = black ; hTauAT = red ) of excitatory postsynaptic field potentials ( fEPSP ) depicts examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale ( s . p . ) of area CA3 in slices in which calcium imaging experiments were conducted . The example depolarization evokes averaged calcium influxes into neurons depicted in the calcium imaging graph . The graph shows the mean of [ Ca + + ] i changes in response to high KCl in stratum radiatum and stratum pyramidale of area CA3 in Fura2 - AM loaded hippocampal slices . After depolarization [ Ca + + ] i rises up to ~ 600 nM in hTauAT slices ( red trace , n = 6 slices , prepared from at least three animals ) , compared to ~ 300 nM in control slices ( black trace , n = 6 slices ; prepared from at least three animals ) . Note that even under resting conditions [ Ca + + ] i is elevated to ~ 120nM due to hTauAT expression . D . Quantification of the maximum [ Ca + + ] i from ratiometric images as depicted in ( A ) and ( B ) after background subtraction and selection of regions of interest ( 10 circles of fixed diameter ) in stratum radiatum border to stratum pyramidale of area CA3 . Under resting conditions [ Ca + + ] i was elevated in transgenic slice cultures ( ~ 120 nM , n = 11 ) compared to controls ( ~ 80 nM , n = 8 ) . Slices were incubated with one of the following channel blockers : Nifedipine ( NIF , VGCC ; Ctrl n = 8 ; A152T : n = 7 ) , APV ( NMDAR ; Ctrl n = 7 ; A152T n = 8 ) , ifenprodil ( IFEN , NMDAR ; Ctrl n = 8 ; A152T n = 7 ) , memantine ( MEM , NMDAR ; Ctrl / A152T n = 6 ) , 6 - cyano - 7 - nitroquinoxaline - 2 , 3 - dione ( CNQX ; AMPAR ; Ctrl n = 8 ; A152T n = 7 ) ) , tetrodotoxin ( TTX , VGNaC ; Ctrl n = 4 ; A152T n = 5 ) , tetanus neurotoxin ( TeNT , neurotransmitter release ; A152T n = 6 ) or in Ca + + free buffer ( Ctrl n = 10 ; A152T n = 7 ) . For each experiment slice preparations from at least three different mice were used . One - way ANOVA followed by Tukey ' s post - hoc test ; Ctrl : F ( 7 / 51 ) . 3 , 533 ; p = 0 . 0036 ; A152T : F ( 8 / 55 ) = 5 , 230 ; p < 0 . 0001 . For an overview of drug concentrations see EV Table 1 . E . Quantification of the maximum peak increase in intracellular calcium in the same slices than in ( D ) after high KCl stimulation . One way ANOVA followed by Tukey ' s post - hoc test Ctrl : F ( 7 / 45 ) = 4 , 080 ; p = 0 . 0015 ; A152T : F ( 8 / 53 ) = 7 , 580 ; p < 0 . 0001 ) . Error bars represent SEM ; One - Way ANOVA * p < 0 . 05 ; * * p < 0 . 01 and * * * p < 0 . 001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Slices (DIV15) from control mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images are presented at baseline and after application of high potassium. Note the increase in intracellular Ca++ ([Ca++]i) after KCl application.B. Slices (DIV15) from hTauAT mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images at baseline and after application of high potassium in area CA3 are depicted. Note the increase in [Ca++]i (false color legend) under both conditions in hTauAT slices compared to controls.C. Electrophysiological example traces (control = black; hTauAT = red) of excitatory postsynaptic field potentials (fEPSP) depicts examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale (s.p.) of area CA3 in slices in which calcium imaging experiments were conducted. The example depolarization evokes averaged calcium influxes into neurons depicted in the calcium imaging graph. The graph shows the mean of [Ca++]i changes in response to high KCl in stratum radiatum and stratum pyramidale of area CA3 in Fura2-AM loaded hippocampal slices. After depolarization [Ca++]i rises up to ~600 nM in hTauAT slices (red trace, n=6 slices, prepared from at least three animals), compared to ~300 nM in control slices (black trace, n=6 slices; prepared from at least three animals). Note that even under resting conditions [Ca++]i is elevated to ~120nM due to hTauAT expression.D. Quantification of the maximum [Ca++]i from ratiometric images as depicted in (A) and (B) after background subtraction and selection of regions of interest (10 circles of fixed diameter) in stratum radiatum border to stratum pyramidale of area CA3. Under resting conditions [Ca++]i was elevated in transgenic slice cultures (~120 nM, n=11) compared to controls (~80 nM, n=8). Slices were incubated with one of the following channel blockers: Nifedipine (NIF, VGCC; Ctrl n=8; A152T: n=7), APV (NMDAR; Ctrl n=7; A152T n=8), ifenprodil (IFEN, NMDAR; Ctrl n=8; A152T n=7), memantine (MEM, NMDAR; Ctrl/A152T n=6), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; AMPAR; Ctrl n=8; A152T n=7)), tetrodotoxin (TTX, VGNaC; Ctrl n=4; A152T n=5), tetanus neurotoxin (TeNT, neurotransmitter release; A152T n=6) or in Ca++ free buffer (Ctrl n=10; A152T n=7). For each experiment slice preparations from at least three different mice were used. One-way ANOVA followed by Tukey's post-hoc test; Ctrl: F(7/51) .3,533; p=0.0036; A152T: F(8/55)=5,230; p<0.0001.For an overview of drug concentrations see EV Table 1.E. Quantification of the maximum peak increase in intracellular calcium in the same slices than in (D) after high KCl stimulation. One way ANOVA followed by Tukey's post-hoc test Ctrl: F(7/45)=4,080; p=0.0015; A152T: F(8/53)=7,580; p<0.0001).Error bars represent SEM; One-Way ANOVA *p<0.05; **p<0.01 and ***p<0.001.