Figure 2 ( a ) Expression of CLV3 , mCherry , TEL2 , PAN , and the meristem marker genes STM and KNAT1 . Asterisks indicate timepoints of significantly different expression ( Wald test , Benjamini & Hochberg corrected , q < 0 . 05 ) between stem and non - stem cells for each time point ( N = 2 ) . + = stem cell nuclei ; - = non - stem cell nuclei , E = nuclei from embryos , D7 / 14 / 35 = nuclei from 7 / 14 / 35 day - old plants , S14 = nuclei from 14 d - old above - ground seedlings . ( c ) Number of DEGs between stem and non - stem cells at each timepoint . The banded portion of the bars indicates the number of transcription factor genes ( also in parenthesis ) .

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Figure 2(a) Expression of CLV3, mCherry, TEL2, PAN, and the meristem marker genes STM and KNAT1. Asterisks indicate timepoints of significantly different expression (Wald test, Benjamini & Hochberg corrected, q<0.05) between stem and non-stem cells for each time point (N = 2). + = stem cell nuclei; - = non-stem cell nuclei, E = nuclei from embryos, D7/14/35 = nuclei from 7/14/35 day-old plants, S14 = nuclei from 14 d-old above-ground seedlings.(c) Number of DEGs between stem and non-stem cells at each timepoint. The banded portion of the bars indicates the number of transcription factor genes (also in parenthesis).


Figure 3 ( b ) Expression of individual significantly up - regulated genes marked with # in ( a ) . Asterisks indicate timepoints of significantly different expression ( Wald test , Benjamini & Hochberg corrected , q < 0 . 05 ) between stem and non - stem cells for each time point ( N = 2 ) . + = stem cells ; - = non - stem cells , E = nuclei from embryos , D7 / 14 / 35 = nuclei from 7 / 14 / 35 day - old plants , S14 = nuclei from 14 d - old above - ground seedlings .

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Figure 3(b) Expression of individual significantly up-regulated genes marked with # in (a). Asterisks indicate timepoints of significantly different expression (Wald test, Benjamini & Hochberg corrected, q<0.05) between stem and non-stem cells for each time point (N = 2).+ = stem cells; - = non-stem cells, E = nuclei from embryos, D7/14/35 = nuclei from 7/14/35 day-old plants, S14 = nuclei from 14 d-old above-ground seedlings.


Figure 4 ( a ) Box plot summarizing differential expression of unambiguously mapped individual TEs , compared with all protein - coding genes , or housekeeping genes . Box plots outline the interquartile range ( IQR ) with the median and whiskers + / - 1 . 5 IQR . For all p - values , see also Appendix Table EV6 . * * indicate a p - value smaller than 1 . 01e - 11 ( two sided non paired Wilcoxon rank sum test ) .

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Figure 4(a) Box plot summarizing differential expression of unambiguously mapped individual TEs, compared with all protein-coding genes, or housekeeping genes. Box plots outline the interquartile range (IQR) with the median and whiskers +/- 1.5 IQR . For all p-values, see also Appendix Table EV6. ** indicate a p-value smaller than 1.01e-11 (two sided non paired Wilcoxon rank sum test).


Figure 5 ( a ) Increased expression of TEs in stem cells of D7 seedlings according to qRT - PCR . Box plots outline the interquartile range ( IQR ) with the median and whiskers + / - 1 . 5 IQR . N = 2 ( ATLANTYS3 , ATCOPIA39 , ATCOPIA49 , ATCOPIA29 , ATGP1 - 1 ) , N = 3 ( VANDAL6 , ATGP2 , ATGP1 - 2 , ATLINE1 , ATGP1 - 3 ) , N = 4 ( ATHILA6A , ATCOPIA83 ) , N = 5 ( VANDAL12 , ATHILA3 , ATCOPIA22 ) , and N = 6 for CLAVATA . * p < 0 . 01 , * * p < 0 . 001 ( t - test ) . ( b ) Heatmap of TE expression in several silencing mutants determined by qRT - PCR relative to a housekeeping gene . To be able to display data in log - scale also for samples with zero expression , a value of 1 . 0e - 6 ( factor 10 smaller than the smallest value ) was added to all values . Data for nuclei and CLV3 : : H2BmCherry are the same as in ( a ) . For seedling samples , N = 2 ( ATCOPIA49 , ATHILA3 ) , N = 3 ( ATCOPIA39 , VANDAL6 , ATGP2 , ATLINE1 , ATCOPIA29 , ATGP1 - 1 ) , and N = 4 ( ATLANTYS3 , VANDAL12 , ATGP1 - 2 , ATHILA6A ) .

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Figure 5(a) Increased expression of TEs in stem cells of D7 seedlings according to qRT-PCR. Box plots outline the interquartile range (IQR) with the median and whiskers +/- 1.5 IQR. N = 2 (ATLANTYS3, ATCOPIA39, ATCOPIA49, ATCOPIA29, ATGP1-1), N = 3 (VANDAL6, ATGP2, ATGP1-2, ATLINE1, ATGP1-3), N = 4 (ATHILA6A, ATCOPIA83), N = 5 (VANDAL12, ATHILA3, ATCOPIA22), and N = 6 for CLAVATA. *p<0.01, **p<0.001 (t-test).(b) Heatmap of TE expression in several silencing mutants determined by qRT-PCR relative to a housekeeping gene. To be able to display data in log-scale also for samples with zero expression, a value of 1.0e-6 (factor 10 smaller than the smallest value) was added to all values. Data for nuclei and CLV3::H2BmCherry are the same as in (a). For seedling samples, N = 2 (ATCOPIA49, ATHILA3), N = 3 (ATCOPIA39, VANDAL6, ATGP2, ATLINE1, ATCOPIA29, ATGP1-1), and N = 4 (ATLANTYS3, VANDAL12, ATGP1-2, ATHILA6A).


Figure 6 ( a ) CG , CHG , and CHH methylation at chromosome 3 in stem and non - stem cells . The inset for CHG methylation shows a magnification of the pericentromeric region .

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Figure 6(a) CG, CHG, and CHH methylation at chromosome 3 in stem and non-stem cells. The inset for CHG methylation shows a magnification of the pericentromeric region.


Figure 7 ( a ) DNA methylation in % at CHG ( left ) and CHH ( right ) sites in TE with increased expression at D7 , compared between developmental stages ( D7 / 14 / 35 = 7 / 14 / 35 day - old plants ) and nuclei of stem ( + ) and non - stem ( - ) cells . ( b ) Genomic location of the TEs ( blue ) from ( a ) on the five chromosomes . Dark grey bars indicate the location of centromeres and blue lines indicate global TE density .

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Figure 7(a) DNA methylation in % at CHG (left) and CHH (right) sites in TE with increased expression at D7, compared between developmental stages (D7/14/35 = 7/14/35 day-old plants) and nuclei of stem (+) and non-stem (-) cells.(b) Genomic location of the TEs (blue) from (a) on the five chromosomes. Dark grey bars indicate the location of centromeres and blue lines indicate global TE density.


Figure 8 ( a ) DNA methylation differences between stem and non - stem cells and their enrichment within DMRs of different epigenetic mutants using permutation tests ( data from Stroud , Greenberg , Feng , Bernatavichute and Jacobsen 2013 ) . Mutants are in alphabetical order according to the gene acronym . Red indicates an enrichment of methylated DNA in the respective context and blue a depletion .

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Figure 8(a) DNA methylation differences between stem and non-stem cells and their enrichment within DMRs of different epigenetic mutants using permutation tests (data from Stroud, Greenberg, Feng, Bernatavichute and Jacobsen 2013). Mutants are in alphabetical order according to the gene acronym. Red indicates an enrichment of methylated DNA in the respective context and blue a depletion.


Figure 1 ( K TUNEL staining of testis sections from WT and KO mice at four months of age ( K ) White dotted line indicates the outline of testis tubules . L ) TUNEL staining of testis sections from WT and KO mice at four months of age with apoptotic cell number per tubule ( L ) . n = 3 in ( L ) .

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Figure 1(K TUNEL staining of testis sections from WT and KO mice at four months of age (K) White dotted line indicates the outline of testis tubules.L) TUNEL staining of testis sections from WT and KO mice at four months of age with apoptotic cell number per tubule (L). n=3 in (L).


Figure 3 ( A ) Immunostaining of SYCP3 and γH2AX in sections from adult WT mice . Arrow and arrowhead indicate diffuse and dot - like γH2AX staining , respectively . White dotted line indicates the outline of testis tubules . ( B ) Proportion of tubules of γH2AX expression patterns . At least 100 tubules of each mouse were counted . n = 4 for WT and n = 5 for KO . ( C ) Average number of spermatocytes with γH2AX expression patterns per tubule . At least 50 tubules of each mouse were counted . n = 3 . ( D - H ) Representative nuclear spreads of WT and KO spermatocytes at leptonema ( D ) , zygonema ( E ) , early pachynema ( F ) , late pachynema ( G ) and diplonema ( H ) . Spermatocytes were immunostained for SYCP3 , γH2AX , and H1t . Magnified views are indicated by dashed areas . Schematic representation shows homolog characteristic revealed by SYCP3 signals at different backgrounds and stages . Arrowheads indicate bivalent stretches lacking SYCP3 signals in KO . ( I ) Proportion statistics of spermatocytes with discontinuous SYCP3 signals . At least 200 cells of each mouse were counted . n = 3 for WT and n = 4 for KO . ( J ) Meiotic stage frequencies in ( D - H ) . At least 200 cells of each mouse were counted . n = 3 .

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Figure 3(A) Immunostaining of SYCP3 and γH2AX in sections from adult WT mice. Arrow and arrowhead indicate diffuse and dot-like γH2AX staining, respectively. White dotted line indicates the outline of testis tubules. (B) Proportion of tubules of γH2AX expression patterns. At least 100 tubules of each mouse were counted. n=4 for WT and n=5 for KO. (C) Average number of spermatocytes with γH2AX expression patterns per tubule. At least 50 tubules of each mouse were counted. n=3.(D-H) Representative nuclear spreads of WT and KO spermatocytes at leptonema (D), zygonema (E), early pachynema (F), late pachynema (G) and diplonema (H). Spermatocytes were immunostained for SYCP3, γH2AX, and H1t. Magnified views are indicated by dashed areas. Schematic representation shows homolog characteristic revealed by SYCP3 signals at different backgrounds and stages. Arrowheads indicate bivalent stretches lacking SYCP3 signals in KO. (I) Proportion statistics of spermatocytes with discontinuous SYCP3 signals. At least 200 cells of each mouse were counted. n=3 for WT and n=4 for KO. (J) Meiotic stage frequencies in (D-H). At least 200 cells of each mouse were counted. n=3.


Figure 4 ( C ) Nuclear - spread spermatocytes from WT and KO mice were immunostained for SYCP3 and MLH1 . Representative images of pachynema are shown . Magnified views are indicated by dashed areas . ( D ) Number of bivalents without an MLH1 focus per pachytene spermatocyte in ( C ) . n = 3 mice . Nor , normal pachynema ; L 1 , pachynema - like 1 ; L 2 , pachynema - like 2 . ( E - H ) Nuclear - spread spermatocytes from WT and KO mice were immunostained for SYCP3 together with RAD51 ( E ) or DMC1 ( G ) and quantification of RAD51 ( F ) or DMC1 ( H ) focus numbers at early zygonema , late zygonema , and pachynema . Representative images of early zygonema are shown . n = 3 mice .

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Figure 4(C) Nuclear-spread spermatocytes from WT and KO mice were immunostained for SYCP3 and MLH1. Representative images of pachynema are shown. Magnified views are indicated by dashed areas. (D) Number of bivalents without an MLH1 focus per pachytene spermatocyte in (C). n=3 mice. Nor, normal pachynema; L 1, pachynema-like 1; L 2, pachynema-like 2.(E-H) Nuclear-spread spermatocytes from WT and KO mice were immunostained for SYCP3 together with RAD51 (E) or DMC1 (G) and quantification of RAD51 (F) or DMC1 (H) focus numbers at early zygonema, late zygonema, and pachynema. Representative images of early zygonema are shown. n=3 mice.


Figure 5 ( A - D ) Representative nuclear spreads of WT and KO spermatocytes at leptonema ( A ) , zygonema ( B ) , pachynema ( C ) and diplonema ( D ) . Spermatocytes were immunostained for REC8 and SYCP3 . REC8 foci distribute along SYCP3 - labeled structures . Magnified views are indicated by dashed areas . ( E ) Inter - REC8 distances along axial elements . Five stretches or homologs were measured and the average distance was calculated for each nucleus . Each point represents one nucleus . The upper panel is the schematic representation for distance measurement . n = 3 mice . ( F ) Western blot analyses for REC8 and SEPARASE in spermatocytes isolated by FACS after Hoechst staining . n = 3 mice . ( G ) Normalized density of REC8 in ( F ) . n = 3 mice .

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Figure 5(A-D) Representative nuclear spreads of WT and KO spermatocytes at leptonema (A), zygonema (B), pachynema (C) and diplonema (D). Spermatocytes were immunostained for REC8 and SYCP3. REC8 foci distribute along SYCP3-labeled structures. Magnified views are indicated by dashed areas.(E) Inter-REC8 distances along axial elements. Five stretches or homologs were measured and the average distance was calculated for each nucleus. Each point represents one nucleus. The upper panel is the schematic representation for distance measurement. n=3 mice.(F) Western blot analyses for REC8 and SEPARASE in spermatocytes isolated by FACS after Hoechst staining. n=3 mice. (G) Normalized density of REC8 in (F). n=3 mice.


Figure 6 ( E ) Western blot analyses for SECURIN and CYCLIN B1 in spermatocytes isolated by FACS after Hoechst staining . n = 3 mice . ( F ) Normalized density of SECURIN and CYCLIN B1 in ( E ) . n = 3 mice .

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Figure 6(E) Western blot analyses for SECURIN and CYCLIN B1 in spermatocytes isolated by FACS after Hoechst staining. n=3 mice. (F) Normalized density of SECURIN and CYCLIN B1 in (E). n=3 mice.


Figure 7 ( F ) Western blot analyses for REC8 and SEPARASE for cells in ( E ) . n = 3 biological replicates . ( G ) Normalized density of REC8 in ( F ) . n = 3 biological replicates . ( H ) Western blot analyses for SECURIN and CYCLIN B1 for cells in ( E ) . n = 3 biological replicates . ( I ) Normalized density of SECURIN and CYCLIN B1 in ( H ) . n = 3 biological replicates .

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Figure 7(F) Western blot analyses for REC8 and SEPARASE for cells in (E). n=3 biological replicates. (G) Normalized density of REC8 in (F). n=3 biological replicates.(H) Western blot analyses for SECURIN and CYCLIN B1 for cells in (E). n=3 biological replicates. (I) Normalized density of SECURIN and CYCLIN B1 in (H). n=3 biological replicates.


Figure 1Expression of Tardbp , total Sort1 and exon 17b Sort1 mRNAs quantified by Q - PCR in primary hippocampal neurons infected with either scrambled or Tardbp shRNA lentiviruses . Values were first normalized to Gapdh mRNA levels , then plotted as average ± SEM relative to the value of scrambled shRNA ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Western blots of cell lysates and culture supernatants showing TDP - 43 and Sortilin expression in primary hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses . Uninfected cultures were used as an additional control . β - actin immunoblotting and Coomasise Blue staining were used to control equal loading of lysates and supernatants , respectively . Molecular weights are indicated in kDa . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses , immunolabled for BDNF ( green ) , SCG2 ( red ) and counterstained with DAPI ( blue ) in orthogonal views ( dashed lines ) showing colocalization of BDNF and SCG2 in the soma ( white arrows ) . Colocalization between BDNF and SCG2 - positive vesicles expressed as percentage of total BDNF immunoreactivity ± SEM in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses ( N = 3 independent experiments , n > 50 individual cells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Colocalization between BDNF and SCG2 - positive vesicles in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses together with either EGFP or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF / SCG2 colocalization expressed as percentage of total BDNF immunoreactivity ( N = 3 independent experiments , n > 50 individual cells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . BDNF secretion induced by KCl treatment assessed by in - situ BDNF ELISA in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * , p < 0 . 05 ; * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . BDNF secretion in cultured hippocampal neurons infected with scrambled ( left ) or Tardbp ( right ) shRNA lentiviruses together with either EGFP , Sort1 shRNA or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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 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 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 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 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 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

Figure 1Expression of Tardbp, total Sort1 and exon 17b Sort1 mRNAs quantified by Q-PCR in primary hippocampal neurons infected with either scrambled or Tardbp shRNA lentiviruses. Values were first normalized to Gapdh mRNA levels, then plotted as average ± SEM relative to the value of scrambled shRNA (N=3 independent experiments; n=3 wells per condition in each experiment). ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Western blots of cell lysates and culture supernatants showing TDP-43 and Sortilin expression in primary hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Uninfected cultures were used as an additional control. β-actin immunoblotting and Coomasise Blue staining were used to control equal loading of lysates and supernatants, respectively. Molecular weights are indicated in kDa. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses, immunolabled for BDNF (green), SCG2 (red) and counterstained with DAPI (blue) in orthogonal views (dashed lines) showing colocalization of BDNF and SCG2 in the soma (white arrows).Colocalization between BDNF and SCG2-positive vesicles expressed as percentage of total BDNF immunoreactivity ±SEM in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses (N=3 independent experiments, n>50 individual cells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Colocalization between BDNF and SCG2-positive vesicles in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF/SCG2 colocalization expressed as percentage of total BDNF immunoreactivity (N=3 independent experiments, n>50 individual cells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.BDNF secretion in cultured hippocampal neurons infected with scrambled (left) or Tardbp (right) shRNA lentiviruses together with either EGFP, Sort1 shRNA or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Figure 2Representative photomicrographs of TDP - 43 ( green ) and NeuN ( red ) immunohistochemistry in hippocampal CA1 of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Arrows denote areas of depleted TDP - 43 immunoreactivity in the mutant . Percentage of TDP - 43 positive cells relative to NeuN cells in CA1 and the remaining hippocampus ( HC [ - CA1 ] ) of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition ; 5 sections per mouse ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . TDP - 43 intensity in TDP - 43 / NeuN double - positive cells in CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition ; 5 sections per mouse ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Expression of Tardbp mRNA in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice quantified by Q - PCR . Results are presented as average ± SEM relative to levels in Tardbpfx / fx mice , after normalization to Gapdh mRNA . ( N = 3 mice per condition ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Western blots of micro - dissected CA1 and the remaining hippocampus showing expression of TDP - 43 , full - length and truncated ( soluble ) Sortilin , and GAPDH in Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Bands corresponding to soluble Sortilin are indicated ( black arrows ) . GAPDH immunoblotting was used to control for equal loading . Molecular weights are indicated in kDa . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . TDP - 43 expression in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SD of densitometry relative to GAPDH ( N = 3 mice per condition ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Expression of exon 17b Sort1 and total Sort1 mRNAs in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice quantified by Q - PCR . Results are presented as average ± SD relative to levels in Tardbpfx / fx mice , after normalization to Gapdh mRNA . ( N = 3 mice per condition ; * * , p < 0 . 01 Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Expression of full - length ( left ) and truncated ( soluble , right ) Sortilin protein in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SD of densitometry relative to GAPDH ( N = 3 mice per condition ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Figure 2Representative photomicrographs of TDP-43 (green) and NeuN (red) immunohistochemistry in hippocampal CA1 of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Arrows denote areas of depleted TDP-43 immunoreactivity in the mutant.Percentage of TDP-43 positive cells relative to NeuN cells in CA1 and the remaining hippocampus (HC [-CA1]) of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.TDP-43 intensity in TDP-43/NeuN double-positive cells in CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Expression of Tardbp mRNA in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice quantified by Q-PCR. Results are presented as average ± SEM relative to levels in Tardbpfx/fx mice, after normalization to Gapdh mRNA. (N=3 mice per condition; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Western blots of micro-dissected CA1 and the remaining hippocampus showing expression of TDP-43, full-length and truncated (soluble) Sortilin, and GAPDH in Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Bands corresponding to soluble Sortilin are indicated (black arrows). GAPDH immunoblotting was used to control for equal loading. Molecular weights are indicated in kDa. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.TDP-43 expression in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD of densitometry relative to GAPDH (N=3 mice per condition; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Expression of exon 17b Sort1 and total Sort1 mRNAs in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice quantified by Q-PCR. Results are presented as average ± SD relative to levels in Tardbpfx/fx mice, after normalization to Gapdh mRNA. (N=3 mice per condition; **, p<0.01 Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Expression of full-length (left) and truncated (soluble, right) Sortilin protein in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD of densitometry relative to GAPDH (N=3 mice per condition; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Figure 3Representative photomicrograph of Golgi staining in hippocampus of a Tardbpfx / fx mouse . Sholl analysis of apical and basal dendritic arbors of CA1 pyramidal neurons from Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition , 20 neurons per mouse ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . High - magnification representative photomicrograph of Golgi staining of the dendritic arbour of a CA1 pyramidal neuron in a Tardbpfx / fx mouse . Black arrows denote selected dendritic spines . Spine density in apical and basal dendrites of CA1 pyramidal neurons of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SD ( N = 3 mice per condition , 15 sections per mouse ; * , p < 0 . 05 ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Representative photomicrographs of a CamKIIaCRE ; Tardbpfx / fx hippocampus after stereotaxic injection of EGFP - expressing lentivirus immunostained for EGFP ( green ) and NeuN ( red ) , and counterstained with DAPI ( blue ) . The injected area within CA1 is indicated in the merged panel . Expression of Tardbp ( left ) , total Sort1 ( center ) and exon 17b Sort1 ( right ) mRNAs quantified by Q - PCR in micro - dissected CA1 of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice after injection of EGFP - expressing lentivirus or Sortilin rescue virus . Results are presented as average ± SEM relative to levels in Tardbpfx / fx mice injected with EGFP virus , after normalization to Gapdh mRNA . ( N = 3 mice per condition ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Spine density in apical dendrites of CA1 pyramidal neurons of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice after injection of EGFP - expressing lentivirus or Sortilin rescue virus . Results are presented as average ± SEM ( N = 3 mice per condition , 15 sections per mouse ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

O B-PANEL_START 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 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 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 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 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

Figure 3Representative photomicrograph of Golgi staining in hippocampus of a Tardbpfx/fx mouse.Sholl analysis of apical and basal dendritic arbors of CA1 pyramidal neurons from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition, 20 neurons per mouse). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.High-magnification representative photomicrograph of Golgi staining of the dendritic arbour of a CA1 pyramidal neuron in a Tardbpfx/fx mouse. Black arrows denote selected dendritic spines.Spine density in apical and basal dendrites of CA1 pyramidal neurons of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD (N=3 mice per condition, 15 sections per mouse; *, p<0.05; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Representative photomicrographs of a CamKIIaCRE;Tardbpfx/fx hippocampus after stereotaxic injection of EGFP-expressing lentivirus immunostained for EGFP (green) and NeuN (red), and counterstained with DAPI (blue). The injected area within CA1 is indicated in the merged panel.Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in micro-dissected CA1 of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice after injection of EGFP-expressing lentivirus or Sortilin rescue virus. Results are presented as average ± SEM relative to levels in Tardbpfx/fx mice injected with EGFP virus, after normalization to Gapdh mRNA. (N=3 mice per condition). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Spine density in apical dendrites of CA1 pyramidal neurons of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice after injection of EGFP-expressing lentivirus or Sortilin rescue virus. Results are presented as average ± SEM (N=3 mice per condition, 15 sections per mouse). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Figure 4Percentage of change in field excitatory postsynaptic potential ( fEPSP ) recorded from hippocampal area CA1 after theta - burst stimulation ( TBS ) in Schaffer collaterals of hippocampal slices from Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average normalised to t = 0 ± SEM ( N = 7 slices from 3 mice per condition ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Percentage fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice stereotactically injected with EGFP - expressing lentivirus or Sortilin rescue virus ( red ) superimposed to the same data shown in panel ( a ) ( black ) . Results are presented as average normalised to t = 0 ± SEM ( N = 7 slices from 3 mice per condition ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Percentage fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice under control conditions ( black ) or with BDNF ( 200ng / ml ) perfused from t = 0 ( green ) . Results are presented as average normalised to t = 0 ± SEM ( N = 7 slices from 3 mice per condition ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Percentage of total time spent in the target quadrant or in the remaining three quadrants of the Barnes maze for Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice at 3 different probe times : 20 - 30 min after first training session ( short - term memory ) and 24h or 14 days after last training session ( long - term or remote memory , respectively ) . Results are presented as average ± SEM ( N = 8 mice per condition ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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 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 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 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

Figure 4Percentage of change in field excitatory postsynaptic potential (fEPSP) recorded from hippocampal area CA1 after theta-burst stimulation (TBS) in Schaffer collaterals of hippocampal slices from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average normalised to t=0 ± SEM (N=7 slices from 3 mice per condition). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Percentage fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice stereotactically injected with EGFP-expressing lentivirus or Sortilin rescue virus (red) superimposed to the same data shown in panel (a) (black). Results are presented as average normalised to t=0 ± SEM (N=7 slices from 3 mice per condition). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Percentage fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice under control conditions (black) or with BDNF (200ng/ml) perfused from t=0 (green). Results are presented as average normalised to t=0 ± SEM (N=7 slices from 3 mice per condition). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Percentage of total time spent in the target quadrant or in the remaining three quadrants of the Barnes maze for Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice at 3 different probe times: 20-30 min after first training session (short-term memory) and 24h or 14 days after last training session (long-term or remote memory, respectively). Results are presented as average ± SEM (N=8 mice per condition; * p < 0.05, ** p < 0.01, *** p < 0.001). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Figure 5Representative photomicrographs of cultured hippocampal neurons immunostained for TDP - 43 ( green ) , FLAG ( red ) and counterstained with DAPI ( blue ) in control conditions ( untransduced ) or infected with lentivirus expressing FLAG - tagged human TDP - 43 12xQN construct . TDP - 43 intensity in cell nuclei of untransduced or TDP - 43 12xQN - infected hippocampal neurons normalized to mean nuclear intensity in untransduced neurons . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . Student ' s t - test ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using Student ' s t - test ( B ) Expression of Tardbp ( left ) , total Sort1 ( center ) and exon 17b Sort1 ( right ) mRNAs quantified by Q - PCR in hippocampal neurons untransduced ( control ) or infected with lentiviruses expressing Tardbp shRNA or human TDP - 43 12xQN constructs . Values were first normalized to Gapdh mRNA levels , then plotted as average ± SEM relative to levels in untransduced neurons ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . 1 - way ANOVA with Tukey ' s post - hoc ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysisBDNF secretion induced by KCl treatment as assessed by in - situ BDNF ELISA in cultured hippocampal neurons untransduced ( control ) or infected with lentiviruses expressing Tardbp shRNA or human TDP - 43 12xQN constructs . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysisBDNF secretion in cultured hippocampal neurons untransduced ( control ) or infected with lentiviruses expressing Tardbp shRNA or human TDP - 43 12xQN constructs together with either EGFP or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc

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 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 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 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 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

Figure 5Representative photomicrographs of cultured hippocampal neurons immunostained for TDP-43 (green), FLAG (red) and counterstained with DAPI (blue) in control conditions (untransduced) or infected with lentivirus expressing FLAG-tagged human TDP-43 12xQN construct.TDP-43 intensity in cell nuclei of untransduced or TDP-43 12xQN-infected hippocampal neurons normalized to mean nuclear intensity in untransduced neurons. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). Student's t-test; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using Student's t-test (B)Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs. Values were first normalized to Gapdh mRNA levels, then plotted as average ± SEM relative to levels in untransduced neurons (N=3 independent experiments; n=3 wells per condition in each experiment). 1-way ANOVA with Tukey's post-hoc; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysisBDNF secretion induced by KCl treatment as assessed by in-situ BDNF ELISA in cultured hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). **, p<0.01; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysisBDNF secretion in cultured hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis


Figure 6Expression of Tardbp ( left ) , total Sort1 ( center ) and exon 17b Sort1 ( right ) mRNAs quantified by Q - PCR in hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated . Absolute levels of mouse Tardbp and human TARDBP mRNAs were determined using a standard curve and added together to assess total Tardbp mRNA levels relative to control uninfected cells after normalization to Gapdh mRNA . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Western blots of cell lysates showing total TDP - 43 expression in primary hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated . The antibody used here detects both mouse and human TDP - 43 proteins . GAPDH immunoblotting was used to control for equal loading . Molecular weights are noted in kDa . Bands corresponding to mouse and human TDP - 43 are indicated . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . BDNF secretion induced by KCl treatment assessed by in - situ BDNF ELISA in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * , p < 0 . 05 ; * * * , p < 0 . 001 Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . BDNF secretion induced by KCl treatment in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs together with either EGFP or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 5 independent experiments ; n = 5 wells per condition in each experiment ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . Expression of TARDBP ( left ) , total SORT1 ( center ) and exon 17b SORT1 ( right ) mRNAs quantified by Q - PCR in human neurons derived from the indicated stem cells carrying either wild type Met337 ( grey bars ) or disease - associated Val337 ( black bars ) TARDBP alleles . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis . BDNF secretion in stem - cell derived human neurons carrying wild type Met337or Val337 TARDBP alleles , infected with proBDNF lentivirus 5 days prior to depolarization by KCl treatment . Results are presented as average ± SEM ( N = 3 independent experiments ; n = 5 wells per condition in each experiment ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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 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 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 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 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

Figure 6Expression of Tardbp (left), total Sort1 (center) and exon 17b Sort1 (right) mRNAs quantified by Q-PCR in hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. Absolute levels of mouse Tardbp and human TARDBP mRNAs were determined using a standard curve and added together to assess total Tardbp mRNA levels relative to control uninfected cells after normalization to Gapdh mRNA. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Western blots of cell lysates showing total TDP-43 expression in primary hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. The antibody used here detects both mouse and human TDP-43 proteins. GAPDH immunoblotting was used to control for equal loading. Molecular weights are noted in kDa. Bands corresponding to mouse and human TDP-43 are indicated. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). *, p<0.05; ***, p<0.001 Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.BDNF secretion induced by KCl treatment in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.Expression of TARDBP (left), total SORT1 (center) and exon 17b SORT1 (right) mRNAs quantified by Q-PCR in human neurons derived from the indicated stem cells carrying either wild type Met337 (grey bars) or disease-associated Val337 (black bars) TARDBP alleles. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.BDNF secretion in stem-cell derived human neurons carrying wild type Met337or Val337 TARDBP alleles, infected with proBDNF lentivirus 5 days prior to depolarization by KCl treatment. Results are presented as average ± SEM (N=3 independent experiments; n=5 wells per condition in each experiment). **, p<0.01; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Figure 1A . Ig sequence diversification in wild type and h3 . 3 . Each line represents an IGVLR gene from a population cells that have expanded for 36 divisions after cloning . Blue bars = gene conversions ; green lollipops = point mutations ; green bars = ambiguous mutations ( Sale et al , 2001 ) . B . Quantification of sequence diversification in wild type and two h3 . 3 clones expressed as unique events ( gene conversions or point mutations ) per kb per generation . ( Sequences analysed : WT n = 142 , for h3 . 3 c20 n = 140 , for h3 . 3 c32 n = 190 ) . C . Quantification of endogenous AID mRNA in wild type and h3 . 3 cells . The graph represents the mean and SD of four independent RNA extractions of wild type and h3 . 3c20 . p value calculated with the unpaired T test . D . Fluctuation analysis for the formation of sIg - loss variants in wild type ( black circles ) and h3 . 3 clones ( black diamonds ) and in two wild type clones ( blue circles ) , two h3 . 3 clones ( blue diamonds ) expressing hAIDup ( Wang et al , 2009 ) at 15 - 20 times the level of wild type AID . The final column shows h3 . 3 cells with hAIDup complemented with H3 . 3 ( blue triangles ) . Clones were expanded for 36 cell divisions at which point the percentage of sIgM negative cells in each population was determined by flow cytometry . Error bars represent mean and 1 S . D . P values calculated with the Mann - Whitney test . E . Ig sequence diversification in wild type , h3 . 3 and complemented h3 . 3 cells overexpressing hAIDup . Key as in ( A ) plus * = deletion or duplication . F . Mutation rate as a function of hAIDup expression in wild type and h3 . 3 . Filled symbols represent clones overexpressing hAIDup , open symbols clones with natural AID expression . Linear regression lines are shown .

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 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 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 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

Figure 1A. Ig sequence diversification in wild type and h3.3. Each line represents an IGVLR gene from a population cells that have expanded for 36 divisions after cloning. Blue bars = gene conversions; green lollipops = point mutations; green bars = ambiguous mutations (Sale et al, 2001).B. Quantification of sequence diversification in wild type and two h3.3 clones expressed as unique events (gene conversions or point mutations) per kb per generation. (Sequences analysed: WT n = 142, for h3.3 c20 n = 140, for h3.3 c32 n = 190).C. Quantification of endogenous AID mRNA in wild type and h3.3 cells. The graph represents the mean and SD of four independent RNA extractions of wild type and h3.3c20. p value calculated with the unpaired T test.D. Fluctuation analysis for the formation of sIg-loss variants in wild type (black circles) and h3.3 clones (black diamonds) and in two wild type clones (blue circles), two h3.3 clones (blue diamonds) expressing hAIDup (Wang et al, 2009) at 15 - 20 times the level of wild type AID. The final column shows h3.3 cells with hAIDup complemented with H3.3 (blue triangles). Clones were expanded for 36 cell divisions at which point the percentage of sIgM negative cells in each population was determined by flow cytometry. Error bars represent mean and 1 S.D. P values calculated with the Mann-Whitney test.E. Ig sequence diversification in wild type, h3.3 and complemented h3.3 cells overexpressing hAIDup. Key as in (A) plus * = deletion or duplication.F. Mutation rate as a function of hAIDup expression in wild type and h3.3. Filled symbols represent clones overexpressing hAIDup, open symbols clones with natural AID expression. Linear regression lines are shown.


Figure 2B . Diversification in wild type and h3 . 3 cells expressing similar levels of hAIDup and UGI . Red lollipops = transitions at C / T ; Black lollipops = other point mutations ; blue bars = gene conversions . C . Mutation rate as a function of hAIDup expression in UGI - expressing wild type and h3 . 3 cells . Each clone was expanded for 36 cell divisions and hAIDup expression determined by qPCR during the last week of culture .

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Figure 2B. Diversification in wild type and h3.3 cells expressing similar levels of hAIDup and UGI. Red lollipops = transitions at C/T; Black lollipops = other point mutations; blue bars = gene conversions.C. Mutation rate as a function of hAIDup expression in UGI-expressing wild type and h3.3 cells. Each clone was expanded for 36 cell divisions and hAIDup expression determined by qPCR during the last week of culture.


Figure 3A . Enrichment of H3 . 3 in the rearranged allele of the Ig light chain ( IGVLR ) compared with the unrearranged allele ( IGVLUR ) . Each bar represents the mean of 3 independent ChIPs each assayed with 3 repeat qPCRs ( * * * * = p < 0 . 0001 , unpaired T - test ) . B . Igλ mRNA levels in wild type and h3 . 3 cells determined by qPCR , normalised to EF - 1α expression and to WT . n = 3 ( ns = not significant , unpaired T - test ) . C . Surface Ig ( sIg ) in multiple clones of wild type ( blue ) and h3 . 3 ( red ) cells . Green = unstained control . D . ChIP for H3K4me3 and H3K36me3 in IGVLR and IGVLUR . Each bar represents the mean of 3 ChIPs for H3K4me3 and IgG and 2 for H3K36me3 , each assayed with 3 qPCR repeats ( statistical analysis as in Panel A ) . E . 4sUDRB mapping of transcription kinetics through IGVL . Representative locations of qPCR amplicons ( blue ) are shown relative to the TSS of IGVL ( distance is shown in parenthesis ) . qPCR signals were normalised to the enrichment at time zero ( before release ) . Error bars represent 1SD of qPCR triplicates .

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 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 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 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

Figure 3A. Enrichment of H3.3 in the rearranged allele of the Ig light chain (IGVLR) compared with the unrearranged allele (IGVLUR). Each bar represents the mean of 3 independent ChIPs each assayed with 3 repeat qPCRs (**** = p<0.0001, unpaired T-test).B. Igλ mRNA levels in wild type and h3.3 cells determined by qPCR, normalised to EF-1α expression and to WT. n = 3 (ns = not significant, unpaired T-test).C. Surface Ig (sIg) in multiple clones of wild type (blue) and h3.3 (red) cells. Green = unstained control.D. ChIP for H3K4me3 and H3K36me3 in IGVLR and IGVLUR. Each bar represents the mean of 3 ChIPs for H3K4me3 and IgG and 2 for H3K36me3, each assayed with 3 qPCR repeats (statistical analysis as in Panel A).E. 4sUDRB mapping of transcription kinetics through IGVL. Representative locations of qPCR amplicons (blue) are shown relative to the TSS of IGVL (distance is shown in parenthesis). qPCR signals were normalised to the enrichment at time zero (before release). Error bars represent 1SD of qPCR triplicates.


Figure 4A . DNA - RNA hybrids in IGVL and IGCL in wild type and h3 . 3 . The DRIP signal is normalised to total input and to wild type IgVλ ( * * * = p < 0 . 001 , ns = not significant , unpaired T - test ) . B . MNase mapping reveals that loss of H3 . 3 does not affect average Igλ nucleosome positioning . The red bars indicate the qPCR amplicons for the MNase mapping . The qPCR signals are normalised to the first amplicon at the TSS . Error bars represent 1SD of 2 experimental repeats . C . DNA - RNA hybrids in IGVL of wild type clones either without overexpression of RNAseHI ( red , clones 1 and 2 , each assayed with three qPCR repeats ) or with overexpression of RNAseHI ( blue , clones 3 and 4 , each assayed with three qPCR repeats ) ( * * * = p < 0 . 001 , * * = p < 0 . 01 ; unpaired T - test ) . Expression of hAIDup - FLAG and chickenRNAseHI - YFP in these clones is shown in Figure EV6 . D . Rate of Ig diversification in wild type cells with and without overexpression of RNAseHI .

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 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 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

Figure 4A. DNA-RNA hybrids in IGVL and IGCL in wild type and h3.3. The DRIP signal is normalised to total input and to wild type IgVλ (*** = p<0.001, ns = not significant, unpaired T-test).B. MNase mapping reveals that loss of H3.3 does not affect average Igλ nucleosome positioning. The red bars indicate the qPCR amplicons for the MNase mapping. The qPCR signals are normalised to the first amplicon at the TSS. Error bars represent 1SD of 2 experimental repeats.C. DNA-RNA hybrids in IGVL of wild type clones either without overexpression of RNAseHI (red, clones 1 and 2, each assayed with three qPCR repeats) or with overexpression of RNAseHI (blue, clones 3 and 4, each assayed with three qPCR repeats) (*** = p<0.001, ** = p < 0.01; unpaired T-test). Expression of hAIDup-FLAG and chickenRNAseHI-YFP in these clones is shown in Figure EV6.D. Rate of Ig diversification in wild type cells with and without overexpression of RNAseHI.


Figure 5A . Location of ssDNA tracts ≥ 8nt in IGVL in wild type ( WT ) and h3 . 3 cells detected by bisulphite sequencing . Data is from three independent experiments , the results from each being shown as black , red and green bars respectively . ssDNA on the coding strand is shown above the marker line ( numbered relative to the TSS at 0 ) and on the template strand below . In the centre , the percentage of G and C bases on the coding strand in a 30 bp rolling average window is shown . B . Quantitation of the bisulphite detection of single stranded DNA in wild type , h3 . 3 and h3 . 3 complemented cells , normalised to wild type . p - values calculated with a  test . Sequences analysed : Wild type n = 322 , h3 . 3n = 320 and h3 . 3 + H3 . 3 n = 95 . C . Bisulphite detection of single stranded DNA in wild type cells with and without RNAseHI overexpression . p - value calculated with a chi ‐ square test . Sequences analysed : RNAseH1 + ve n = 94 , RNAseH1 - ve n = 90 .

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 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

Figure 5A. Location of ssDNA tracts ≥ 8nt in IGVL in wild type (WT) and h3.3 cells detected by bisulphite sequencing. Data is from three independent experiments, the results from each being shown as black, red and green bars respectively. ssDNA on the coding strand is shown above the marker line (numbered relative to the TSS at 0) and on the template strand below. In the centre, the percentage of G and C bases on the coding strand in a 30 bp rolling average window is shown.B. Quantitation of the bisulphite detection of single stranded DNA in wild type, h3.3 and h3.3 complemented cells, normalised to wild type. p-values calculated with a  test. Sequences analysed: Wild type n = 322, h3.3n = 320 and h3.3 + H3.3 n = 95.C. Bisulphite detection of single stranded DNA in wild type cells with and without RNAseHI overexpression. p-value calculated with a chi‐square test. Sequences analysed: RNAseH1+ve n = 94, RNAseH1-ve n = 90.


Figure 1A . Relative levels of OMA1 mRNA in colorectal cancer tissues ( n = 105 ) compared with adjacent normal tissues ( n = 43 ) from colorectal cancer patients were shown ( using the GEO dataset GSE21510 ) . The data are presented as mean ± SEM , and statistical significance was determined by a Mann - Whitney test . * * * p < 0 . 001 . B . Kaplan - Meier curves were constructed to analyze and compare between patients with high and low levels of OMA1 in colorectal cancer samples from the GEO dataset GSE17537 . " Low " indicates patients with OMA1 mRNA levels less than the median . " High " indicates patients with OMA1 mRNA levels greater than the median . Statistical analysis was performed using log - rank tests , n = 55 , p = 0 . 0180 . C . The lysates of tumors ( T ) and adjacent normal ( N ) tissues from colorectal cancer patients were analyzed by Western blotting with antibodies against OPA1 or Tubulin . The " c , d and e " bands of OPA1 indicate cleaved OPA1 bands . Tubulin was used as a loading control . D . Control and OMA1 KO HCT116 cells were cultured in hypoxia ( 1 % O2 ) for 0 ( normoxia ) , 24 , or 48 hours . Cell lysates were then assessed by Western blotting with antibodies against OPA1 , OMA1 , or β - Actin . β - Actin was used as a loading control . Representative immunoblots were from n = 3 independent experiments . E . OPA1 - null mouse embryonic fibroblasts ( MEFs ) expressing control ( empty vector ) or OPA1 isoform 1 were cultured in normoxia or hypoxia ( 1 % O2 ) for 24 hours . Cell lysates were analyzed by Western blotting with anti - OPA1 or anti - Tubulin antibody . Representative immunoblots were from n = 3 independent experiments . F . WT , OMA1 KO and OMA1 KO HCT116 cells expressing WT - OMA1 ( OMA1 - Flag ) or proteolytic inactive OMA1 ( E324Q - Flag ) were cultured in hypoxia ( 1 % O2 ) for 0 , 24 , or 48 hours , and the cell lysates were assessed by western blot with antibodies against OMA1 , and GAPDH . β - Actin was used as a loading control ( representative data from three independent experiments ) . The asterisk indicates a nonspecific band . G . The relative protein levels were evaluated by densitometry analysis using ImageJ software and were quantified for the ratio of OMA1 - Flag / β - Actin or E324Q - Flag / β - Actin in hypoxia for 0 , 24 or 48 hours ( n = 3 independent experiments ) . The data are presented as mean ± SD .

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Figure 1A. Relative levels of OMA1 mRNA in colorectal cancer tissues (n=105) compared with adjacent normal tissues (n=43) from colorectal cancer patients were shown (using the GEO dataset GSE21510). The data are presented as mean ± SEM, and statistical significance was determined by a Mann-Whitney test. ***p < 0.001.B. Kaplan-Meier curves were constructed to analyze and compare between patients with high and low levels of OMA1 in colorectal cancer samples from the GEO dataset GSE17537. "Low" indicates patients with OMA1 mRNA levels less than the median. "High" indicates patients with OMA1 mRNA levels greater than the median. Statistical analysis was performed using log-rank tests, n = 55, p=0.0180.C. The lysates of tumors (T) and adjacent normal (N) tissues from colorectal cancer patients were analyzed by Western blotting with antibodies against OPA1 or Tubulin. The "c, d and e" bands of OPA1 indicate cleaved OPA1 bands. Tubulin was used as a loading control.D. Control and OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0 (normoxia), 24, or 48 hours. Cell lysates were then assessed by Western blotting with antibodies against OPA1, OMA1, or β-Actin. β-Actin was used as a loading control. Representative immunoblots were from n = 3 independent experiments.E. OPA1-null mouse embryonic fibroblasts (MEFs) expressing control (empty vector) or OPA1 isoform 1 were cultured in normoxia or hypoxia (1% O2) for 24 hours. Cell lysates were analyzed by Western blotting with anti-OPA1 or anti-Tubulin antibody. Representative immunoblots were from n = 3 independent experiments.F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were cultured in hypoxia (1% O2) for 0, 24, or 48 hours, and the cell lysates were assessed by western blot with antibodies against OMA1, and GAPDH. β-Actin was used as a loading control (representative data from three independent experiments). The asterisk indicates a nonspecific band. G. The relative protein levels were evaluated by densitometry analysis using ImageJ software and were quantified for the ratio of OMA1-Flag/β-Actin or E324Q-Flag/β-Actin in hypoxia for 0, 24 or 48 hours (n = 3 independent experiments). The data are presented as mean ± SD.


Figure 2B . Body weight changes of WT ( n = 11 ) and OMA1 - / - ( n = 12 ) mice during AOM / DSS treatment . Data are presented as mean ± SEM . Statistical significance was assessed by a two - way ANOVA , * * p < 0 . 01 . C . Typical intestine images on day 105 after mice were euthanized . D . Colorectal lengths ( C ) of WT ( n = 11 biological replicates ) and OMA1 - / - ( n = 12 biological replicates ) mice were measured . Data with error bars are presented as mean ± SEM . Statistical significance was assessed by unpaired student ' s t - test , * p < 0 . 05 . E . Representative images of colorectal tumors from WT ( left ) and OMA1 - / - ( right ) mice . F . The number of tumors in WT ( n = 11 ) and OMA1 - / - ( n = 12 ) mice was measured . Each dot represents the tumor number of one individual mouse . Data with error bars are presented as mean ± SEM . Statistical significance was assessed by unpaired student ' s t - test , * * * p < 0 . 001 . G , H . The number of tumors of different sizes ( diameter ) in each mouse ( G ) and the distribution of different tumor sizes ( H ) were shown ( WT n = 11 biological replicates , and OMA1 - / - n = 12 biological replicates ) . Results are shown as mean ± SEM . p - values were calculated by unpaired student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 . I . Histological analysis of colorectal tumors of WT and OMA1 - / - mice was shown by hematoxylin and eosin ( H & E ) staining . J . Relative mRNA expression levels of pro - inflammatory genes ( TNFα , IL - 6 , COX - 2 , CCL2 ) in colorectal homogenates of WT and OMA1 _ KO mice treated with AOM / DSS ( n = 4 - 5 per genotype ) . The data represent the mean ± SEM . , n = 3 independent experiments , and statistical significance was determined by a two - tailed student ' s t - test . * p < 0 . 05 , * * p < 0 . 01 . K . The colorectal tissues of WT and OMA1 - / - mice were stained with Ki67 .

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Figure 2B. Body weight changes of WT (n=11) and OMA1-/- (n=12) mice during AOM/DSS treatment. Data are presented as mean ± SEM. Statistical significance was assessed by a two-way ANOVA, **p < 0.01.C. Typical intestine images on day 105 after mice were euthanized. D. Colorectal lengths (C) of WT (n=11 biological replicates) and OMA1-/- (n=12 biological replicates) mice were measured. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired student's t-test, *p < 0.05. E. Representative images of colorectal tumors from WT (left) and OMA1-/- (right) mice. F. The number of tumors in WT (n=11) and OMA1-/- (n=12) mice was measured. Each dot represents the tumor number of one individual mouse. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired student's t-test, ***p < 0.001. G, H. The number of tumors of different sizes (diameter) in each mouse (G) and the distribution of different tumor sizes (H) were shown (WT n=11 biological replicates, and OMA1-/- n=12 biological replicates). Results are shown as mean ± SEM. p-values were calculated by unpaired student's t-test, **p < 0.01, ***p < 0.001.I. Histological analysis of colorectal tumors of WT and OMA1-/- mice was shown by hematoxylin and eosin (H&E) staining.J. Relative mRNA expression levels of pro-inflammatory genes (TNFα, IL-6, COX-2, CCL2) in colorectal homogenates of WT and OMA1_KO mice treated with AOM/DSS (n =4-5 per genotype). The data represent the mean ± SEM., n = 3 independent experiments, and statistical significance was determined by a two-tailed student's t-test. *p < 0.05, **p < 0.01.K. The colorectal tissues of WT and OMA1-/- mice were stained with Ki67.


Figure 3A , B . Control or OMA1 KO HCT116 cells expressing control ( empty vector ) or OMA1 - Flag were maintained in normoxia or hypoxia ( 1 % O2 ) for 24 hours . The glucose uptake ( A ) and lactate production ( B ) were measured by using microplate reader , and the values were normalized to the protein concentration . Error bars indicate the mean ± SD of three independent experiments , statistical significance was assessed by a two - way ANOVA , * P < 0 . 05 . Cells were lysed and assessed by western blot with antibodies against OMA1 and GAPDH ( A ) . The asterisk indicates a nonspecific band . C . Control and OMA1 KO HCT116 cells were treated with DMSO or 1mM DMOG in fresh medium for 12 h . Subsequent injections of glucose , oligomycin ( Oligo ) , and 2 - deoxy - glucose ( 2 - DG ) were performed as indicated . The extracellular acidification rates ( ECAR ) were measured with a Seahorse Extracellular Flux Analyzer XF96 . The data are presented as mean ± SD ( n = 3 independent experiments ) . D . Quantitative analysis of basal and maximal glycolytic capacity . Error bars are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a two - way ANOVA . N . S . , not significant , * * * p < 0 . 001 . E , F . Lactate production ( E ) and ATP production ( F ) were measured in control or OPA1 KO HCT116 cells upon DMSO or 2 - DG treatment . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . N . S . , not significant , * * p < 0 . 01 , * * * p < 0 . 001 . G , H . Control or OPA1 KO HCT116 cells were maintained in normoxia or hypoxia ( 1 % O2 ) for 24 hours . The glucose uptake ( G ) and lactate production ( H ) were measured , and the values were normalized to the protein concentration . Statistical significance was assessed by a two - way ANOVA ; error bars are presented as mean ± SD of three independent experiments ; N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 .

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 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 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 O O O O O O O O O O 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, B. Control or OMA1 KO HCT116 cells expressing control (empty vector) or OMA1-Flag were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (A) and lactate production (B) were measured by using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD of three independent experiments, statistical significance was assessed by a two-way ANOVA, *P < 0.05. Cells were lysed and assessed by western blot with antibodies against OMA1 and GAPDH (A). The asterisk indicates a nonspecific band.C. Control and OMA1 KO HCT116 cells were treated with DMSO or 1mM DMOG in fresh medium for 12 h. Subsequent injections of glucose, oligomycin (Oligo), and 2-deoxy-glucose (2-DG) were performed as indicated. The extracellular acidification rates (ECAR) were measured with a Seahorse Extracellular Flux Analyzer XF96. The data are presented as mean ± SD (n = 3 independent experiments).D. Quantitative analysis of basal and maximal glycolytic capacity. Error bars are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-way ANOVA. N.S., not significant, ***p < 0.001.E, F. Lactate production (E) and ATP production (F) were measured in control or OPA1 KO HCT116 cells upon DMSO or 2-DG treatment. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, **p < 0.01, ***p < 0.001.G, H. Control or OPA1 KO HCT116 cells were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (G) and lactate production (H) were measured, and the values were normalized to the protein concentration. Statistical significance was assessed by a two-way ANOVA; error bars are presented as mean ± SD of three independent experiments; N.S., not significant, *p < 0.05, **p < 0.01.


Figure 4A . Control and OMA1 KO HCT116 cells were cultured in normoxia or in hypoxia ( 1 % O2 ) for 24 or 48 hours . The whole cell lysates were analyzed by Western blotting with antibodies against HIF - 1α , HK2 , LDHA , PKM2 , GPI , OMA1 , or β - Actin . The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B . Control or OMA1 KO Hela cells were untreated ( - ) or CoCl2 - treated ( + ) for 24 hours with 10 mM MG132 or vehicle added for the last 4 hours . Cell lysates were assessed by Western blotting with antibodies against HIF - 1α or β - Actin . Relative protein levels were further evaluated by densitometry analysis . Error bars indicate the mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 . C . Dihydroethidium ( DHE ) staining of optimum cutting temperature ( OCT ) covered tissues from the colons of WT and OMA1 - / - mice ; DAPI was used as counterstain . DHE was shown in red color , while DAPI in blue color ( representative data from three independent experiments ) . D . Quantification of DHE staining . Error bars are presented as mean ± SD ( n = 3 biological replicates ) , statistical significance was assessed by a two - way ANOVA , * * p < 0 . 01 , * * * P < 0 . 001 . E . Control or OMA1 KO HCT116 cells cultured in normoxia or hypoxia ( 1 % O2 ) for 12 hours were stained with mitoSOX , and then analyzed by confocal microscopy ( representative data from three independent experiments ) . F . The fluorescence intensity of mitoSOX was further analyzed by ImageJ software . Bars indicate the mean ± SD of three independent experiments by a two - way ANOVA , * * * p < 0 . 001 . G - I . Control or OMA1 _ KO HCT116 cells were cultured in normoxia or hypoxia for 12 hours , and were treated with DMSO , NAC ( 10 mM ) or Rotenone ( 1 μM ) for 12 hours at the same time . The whole cell lysates were analyzed by Western blotting with antibodies against HIF - 1α , and Tubulin ( G ) . The glucose uptake ( H ) and lactate production ( I ) were measured using microplate reader , and the values were normalized to the protein concentration . Error bars indicate the mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by two - way ANOVA . N . S . , not significant , * P < 0 . 05 , * * * 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 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 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 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 O O O O O O O O O O O

Figure 4A. Control and OMA1 KO HCT116 cells were cultured in normoxia or in hypoxia (1% O2) for 24 or 48 hours. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, HK2, LDHA, PKM2, GPI, OMA1, or β-Actin. The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.B. Control or OMA1 KO Hela cells were untreated (-) or CoCl2-treated (+) for 24 hours with 10 mM MG132 or vehicle added for the last 4 hours. Cell lysates were assessed by Western blotting with antibodies against HIF-1α or β-Actin. Relative protein levels were further evaluated by densitometry analysis. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01.C. Dihydroethidium (DHE) staining of optimum cutting temperature (OCT) covered tissues from the colons of WT and OMA1-/- mice; DAPI was used as counterstain. DHE was shown in red color, while DAPI in blue color (representative data from three independent experiments). D. Quantification of DHE staining. Error bars are presented as mean ± SD (n=3 biological replicates), statistical significance was assessed by a two-way ANOVA, **p < 0.01, ***P< 0.001. E. Control or OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours were stained with mitoSOX, and then analyzed by confocal microscopy (representative data from three independent experiments). F. The fluorescence intensity of mitoSOX was further analyzed by ImageJ software. Bars indicate the mean ± SD of three independent experiments by a two-way ANOVA, ***p < 0.001. G-I. Control or OMA1_KO HCT116 cells were cultured in normoxia or hypoxia for 12 hours, and were treated with DMSO, NAC (10 mM) or Rotenone (1 μM) for 12 hours at the same time. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, and Tubulin (G). The glucose uptake (H) and lactate production (I) were measured using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-way ANOVA. N.S., not significant, *P < 0.05, ***P< 0.001.