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

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(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.


( F ) Western blot analyses for REC8 SEPARASE for in ( E ) . n = 3 biological replicates . ( G ) Normalized density of REC8 in ( F ) . n = 3 biological replicates .

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(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 CYCLIN B1 for in ( E ) . n = 3 biological replicates . ( I ) Normalized density of SECURIN CYCLIN B1 in ( H ) . 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.


Expression of Tardbp , total Sort1 exon 17b Sort1 mRNAs quantified by Q - PCR in hippocampal neurons infected with either scrambled or Tardbp shRNA lentiviruses . Values were first normalized to Gapdh mRNA , then plotted as average ± relative to 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

Expression 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 lysates culture supernatants showing TDP - 43 Sortilin expression in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses . Uninfected cultures were used as an additional control . β - actin immunoblotting Coomasise Blue were used to control equal loading of lysates supernatants , respectively . Molecular weights are indicated in kDa . Data information : Results are presented as average ± standard error of mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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 ) counterstained with DAPI ( blue ) in orthogonal views ( dashed lines ) showing colocalization of BDNF SCG2 in soma ( arrows ) .

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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 SCG2 - positive vesicles expressed as of total BDNF immunoreactivity ± in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses ( N = 3 independent experiments , n > 50 per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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 SCG2 - positive vesicles in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses together with either EGFP or Sortilin rescue . Histogram bars show average ± BDNF / SCG2 colocalization expressed as of total BDNF immunoreactivity ( N = 3 independent experiments , n > 50 per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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 ± BDNF 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 . Histogram bars show average ± BDNF 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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.


Representative photomicrographs of TDP - 43 ( green ) NeuN ( red ) immunohistochemistry in hippocampal CA1 of Tardbpfx / fx CamKIIaCRE ; Tardbpfx / fx mice . Arrows denote areas of depleted TDP - 43 immunoreactivity in mutant .

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Representative 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.


of TDP - 43 positive relative to NeuN in CA1 remaining hippocampus ( HC [ - CA1 ] ) of Tardbpfx / fx CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± ( N = 3 mice per condition ; 5 sections per mouse ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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 in TDP - 43 / NeuN double - positive in CA1 remaining hippocampus of Tardbpfx / fx CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± ( 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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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 remaining hippocampus of Tardbpfx / fx CamKIIaCRE ; Tardbpfx / fx mice quantified by Q - PCR . Results are presented as average ± relative to 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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 remaining hippocampus showing expression of TDP - 43 , full - truncated ( soluble ) Sortilin , GAPDH in Tardbpfx / fx 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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 remaining hippocampus of Tardbpfx / fx 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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 total Sort1 mRNAs in micro - dissected CA1 remaining hippocampus of Tardbpfx / fx CamKIIaCRE ; Tardbpfx / fx mice quantified by Q - PCR . Results are presented as average ± SD relative to 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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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 - ( left ) truncated ( soluble , right ) Sortilin in micro - dissected CA1 remaining hippocampus of Tardbpfx / fx 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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.


Representative photomicrograph of Golgi in hippocampus of a Tardbpfx / fx mouse .

Representative photomicrograph of Golgi staining in hippocampus of a Tardbpfx/fx mouse.


Sholl of apical basal dendritic arbors of CA1 pyramidal neurons from Tardbpfx / fx CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± ( 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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 of dendritic arbour of a CA1 pyramidal neuron in a Tardbpfx / fx mouse . Black arrows denote selected dendritic spines .

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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 basal dendrites of CA1 pyramidal neurons of Tardbpfx / fx 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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 ) NeuN ( red ) , counterstained with DAPI ( blue ) . injected within CA1 is indicated in merged panel .

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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 ) exon 17b Sort1 ( right ) mRNAs quantified by Q - PCR in micro - dissected CA1 of Tardbpfx / fx CamKIIaCRE ; Tardbpfx / fx mice after injection of EGFP - expressing lentivirus or Sortilin rescue . Results are presented as average ± relative to in Tardbpfx / fx mice injected with EGFP , after normalization to Gapdh mRNA . ( N = 3 mice per condition ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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 CamKIIaCRE ; Tardbpfx / fx mice after injection of EGFP - expressing lentivirus or Sortilin rescue . Results are presented as average ± ( N = 3 mice per condition , 15 sections per mouse ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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.


of change in field excitatory postsynaptic potential ( fEPSP ) recorded from hippocampal CA1 after theta - burst stimulation ( TBS ) in Schaffer collaterals of hippocampal slices from Tardbpfx / fx CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average normalised to t = 0 ± ( 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

Percentage 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.


fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx / fx CamKIIaCRE ; Tardbpfx / fx mice stereotactically injected with EGFP - expressing lentivirus or Sortilin rescue ( red ) superimposed to same data shown in panel ( a ) ( black ) . Results are presented as average normalised to t = 0 ± ( 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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.


fEPSP change in CA1 after TBS in Schaffer collaterals of hippocampal slices from Tardbpfx / fx 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 ± ( 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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.


of total time spent in target quadrant or in remaining three quadrants of Barnes maze for Tardbpfx / fx CamKIIaCRE ; Tardbpfx / fx mice at 3 different probe times : 20 - 30 min after first training session ( short - term memory ) 24h or 14 days after last training session ( long - term or remote memory , respectively ) . Results are presented as average ± ( N = 8 mice per condition ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data information : Results are presented as average ± standard error of mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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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.


Representative photomicrographs of cultured hippocampal neurons immunostained for TDP - 43 ( green ) , FLAG ( red ) counterstained with DAPI ( blue ) in control conditions ( untransduced ) or infected with lentivirus expressing FLAG - tagged human TDP - 43 12xQN construct .

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Representative 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 in nuclei of untransduced or TDP - 43 12xQN - infected hippocampal neurons normalized to mean in untransduced neurons . Results are presented as average ± ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . Student ' s t - ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of mean ( ) . statistical were performed using Student ' s t - ( B )

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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 ) 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 , then plotted as average ± relative to 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc

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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 analysis


BDNF 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 ± BDNF 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BDNF 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 analysis


BDNF 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 . Histogram bars show average ± BDNF 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc

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BDNF 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


Expression of Tardbp ( left ) , total Sort1 ( center ) exon 17b Sort1 ( right ) mRNAs quantified by Q - PCR in hippocampal neurons infected with Tardbp shRNA mutant human TARDBP lentivirus constructs as indicated . Absolute of mouse Tardbp human TARDBP mRNAs were determined using a standard curve added together to assess total Tardbp mRNA relative to control uninfected after normalization to Gapdh mRNA . Results are presented as average ± ( 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

Expression 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 lysates showing total TDP - 43 expression in hippocampal neurons infected with Tardbp shRNA mutant human TARDBP lentivirus constructs as indicated . antibody used here detects both mouse human TDP - 43 . GAPDH immunoblotting was used to control for equal loading . Molecular weights are noted in kDa . Bands corresponding to mouse human TDP - 43 are indicated . Data information : Results are presented as average ± standard error of mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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

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 mutant human TARDBP lentivirus constructs as indicated . Histogram bars show average ± BDNF 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 mutant human TARDBP lentivirus constructs together with either EGFP or Sortilin rescue . Histogram bars show average ± BDNF 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) exon 17b SORT1 ( right ) mRNAs quantified by Q - PCR in human neurons derived from indicated carrying either wild type Met337 ( grey bars ) or disease - associated Val337 ( black bars ) TARDBP alleles . Results are presented as average ± ( 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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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 - 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 ± ( 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 mean ( ) . statistical were performed using 2 - way ANOVA with Sidak post hoc .

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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.


A . Ig diversification in wild type h3 . 3 . Each represents an IGVLR gene from a population 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 . of diversification in wild type 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 ) .

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A. 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 . of endogenous AID mRNA in wild type h3 . 3 . graph represents mean SD of four independent extractions of wild type h3 . 3c20 . p value calculated with unpaired T .

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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 for formation of sIg - loss variants in wild type ( black circles ) h3 . 3 clones ( black diamonds ) in two wild type clones ( blue circles ) , two h3 . 3 clones ( blue diamonds ) expressing hAIDup ( Wang et al , 2009 ) at 15 - 20 times level of wild type AID . final column shows h3 . 3 with hAIDup complemented with H3 . 3 ( blue triangles ) . Clones were expanded for 36 divisions at which point of sIgM negative in each population was determined by flow cytometry . Error bars represent mean 1 S . D . P values calculated with Mann - Whitney .

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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 diversification in wild type , h3 . 3 complemented h3 . 3 overexpressing hAIDup . Key as in ( A ) plus * = deletion or duplication .

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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 h3 . 3 . Filled symbols represent clones overexpressing hAIDup , open symbols clones with natural AID expression . Linear regression lines are shown .

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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.


B . Diversification in wild type h3 . 3 expressing similar of hAIDup UGI . Red lollipops = transitions at C / T ; Black lollipops = other point mutations ; blue bars = gene conversions .

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B. 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 h3 . 3 . Each clone was expanded for 36 divisions hAIDup expression determined by qPCR during last week of culture .

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.


A . Enrichment of H3 . 3 in rearranged allele of Ig chain ( IGVLR ) compared with unrearranged allele ( IGVLUR ) . Each bar represents mean of 3 independent ChIPs each assayed with 3 repeat qPCRs ( * * * * = p < 0 . 0001 , unpaired T - ) .

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A. 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 in wild type h3 . 3 determined by qPCR , normalised to EF - 1α expression to WT . n = 3 ( ns = not significant , unpaired T - ) .

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 ) h3 . 3 ( red ) . Green = unstained control .

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C. Surface Ig (sIg) in multiple clones of wild type (blue) and h3.3 (red) cells. Green = unstained control.


D . ChIP for H3K4me3 H3K36me3 in IGVLR IGVLUR . Each bar represents mean of 3 ChIPs for H3K4me3 IgG 2 for H3K36me3 , each assayed with 3 qPCR repeats ( statistical as in Panel A ) .

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 TSS of IGVL ( is shown in parenthesis ) . qPCR signals were normalised to enrichment at time zero ( before release ) . Error bars represent 1SD of qPCR triplicates .

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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.


A . - hybrids in IGVL IGCL in wild type h3 . 3 . DRIP signal is normalised to total input to wild type IgVλ ( * * * = p < 0 . 001 , ns = not significant , unpaired T - ) .

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A. 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 . red bars indicate qPCR amplicons for MNase mapping . qPCR signals are normalised to first amplicon at TSS . Error bars represent 1SD of 2 experimental repeats .

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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 . - hybrids in IGVL of wild type clones either without overexpression of RNAseHI ( red , clones 1 2 , each assayed with three qPCR repeats ) or with overexpression of RNAseHI ( blue , clones 3 4 , each assayed with three qPCR repeats ) ( * * * = p < 0 . 001 , * * = p < 0 . 01 ; unpaired T - ) . Expression of hAIDup - FLAG chickenRNAseHI - YFP in these clones is shown in Figure EV6 .

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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 with without overexpression of RNAseHI .

D. Rate of Ig diversification in wild type cells with and without overexpression of RNAseHI.


A . Location of ssDNA tracts ≥ 8nt in IGVL in wild type ( WT ) h3 . 3 detected by bisulphite sequencing . Data is from three independent experiments , results from each being shown as black , red green bars respectively . ssDNA on coding strand is shown above marker ( numbered relative to TSS at 0 ) on template strand below . In centre , of G C bases on coding strand in a 30 bp rolling average window is shown . B . Quantitation of bisulphite detection of single stranded in wild type , h3 . 3 h3 . 3 complemented , normalised to wild type . p - values calculated with a  . Sequences analysed : Wild type n = 322 , h3 . 3n = 320 h3 . 3 + H3 . 3 n = 95 .

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A. 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 in wild type with without RNAseHI overexpression . p - value calculated with a chi ‐ square . Sequences analysed : RNAseH1 + ve n = 94 , RNAseH1 - ve n = 90 .

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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.


A . Relative of OMA1 mRNA in colorectal cancer tissues ( n = 105 ) compared with adjacent normal tissues ( n = 43 ) from colorectal cancer were shown ( using GEO dataset GSE21510 ) . data are presented as mean ± , statistical significance was determined by a Mann - Whitney . * * * p < 0 . 001 .

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A. 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 compare between with high low of OMA1 in colorectal cancer samples from GEO dataset GSE17537 . " Low " indicates with OMA1 mRNA less than median . " High " indicates with OMA1 mRNA greater than median . Statistical was performed using log - rank , n = 55 , p = 0 . 0180 .

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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 . lysates of tumors ( T ) adjacent normal ( N ) tissues from colorectal cancer were analyzed by Western blotting with antibodies against OPA1 or Tubulin . " c , d e " bands of OPA1 indicate OPA1 bands . Tubulin was used as a loading control .

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

C. 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 OMA1 KO HCT116 were cultured in hypoxia ( 1 % O2 ) for 0 ( normoxia ) , 24 , or 48 hours . 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 .

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 . lysates were analyzed by Western blotting with anti - OPA1 or anti - Tubulin antibody . Representative immunoblots were from n = 3 independent experiments .

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

E. 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 OMA1 KO HCT116 expressing WT - OMA1 ( OMA1 - Flag ) or proteolytic inactive OMA1 ( E324Q - Flag ) were cultured in hypoxia ( 1 % O2 ) for 0 , 24 , or 48 hours , lysates were assessed by western blot with antibodies against OMA1 , GAPDH . β - Actin was used as a loading control ( representative data from three independent experiments ) . asterisk indicates a nonspecific band . G . relative were evaluated by densitometry using ImageJ software were quantified for of OMA1 - Flag / β - Actin or E324Q - Flag / β - Actin in hypoxia for 0 , 24 or 48 hours ( n = 3 independent experiments ) . data are presented as mean ± SD .

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

F. 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.


B . changes of WT ( n = 11 ) OMA1 - / - ( n = 12 ) mice during AOM / DSS treatment . Data are presented as mean ± . Statistical significance was assessed by a two - way ANOVA , * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 on day 105 after mice were euthanized . D . Colorectal lengths ( C ) of WT ( n = 11 biological replicates ) OMA1 - / - ( n = 12 biological replicates ) mice were measured . Data with error bars are presented as mean ± . Statistical significance was assessed by unpaired student ' s t - , * p < 0 . 05 .

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

C. 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 of colorectal tumors from WT ( left ) OMA1 - / - ( right ) mice . F . number of tumors in WT ( n = 11 ) OMA1 - / - ( n = 12 ) mice was measured . Each dot represents number of one mouse . Data with error bars are presented as mean ± . Statistical significance was assessed by unpaired student ' s t - , * * * p < 0 . 001 .

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

E. 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 . number of tumors of different sizes ( diameter ) in each mouse ( G ) distribution of different sizes ( H ) were shown ( WT n = 11 biological replicates , OMA1 - / - n = 12 biological replicates ) . Results are shown as mean ± . p - values were calculated by unpaired student ' s t - , * * p < 0 . 01 , * * * 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 of colorectal tumors of WT OMA1 - / - mice was shown by hematoxylin eosin ( H & E ) .

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

I. Histological analysis of colorectal tumors of WT and OMA1-/- mice was shown by hematoxylin and eosin (H&E) staining.


J . Relative mRNA expression of pro - inflammatory genes ( TNFα , IL - 6 , COX - 2 , CCL2 ) in colorectal homogenates of WT OMA1 _ KO mice treated with AOM / DSS ( n = 4 - 5 per genotype ) . data represent mean ± . , n = 3 independent experiments , statistical significance was determined by a two - student ' s t - . * p < 0 . 05 , * * p < 0 . 01 .

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

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 . colorectal tissues of WT OMA1 - / - mice were stained with Ki67 .

K. The colorectal tissues of WT and OMA1-/- mice were stained with Ki67.


A , B . Control or OMA1 KO HCT116 expressing control ( empty vector ) or OMA1 - Flag were maintained in normoxia or hypoxia ( 1 % O2 ) for 24 hours . glucose uptake ( A ) lactate production ( B ) were measured by using microplate reader , values were normalized to . Error bars indicate mean ± SD of three independent experiments , statistical significance was assessed by a two - way ANOVA , * P < 0 . 05 . were lysed assessed by western blot with antibodies against OMA1 GAPDH ( A ) . asterisk indicates a nonspecific band .

O O O O O O O O O O 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-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. 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 OMA1 KO HCT116 were treated with DMSO or 1mM DMOG in fresh medium for 12 h . Subsequent injections of glucose , oligomycin ( Oligo ) , 2 - deoxy - glucose ( 2 - DG ) were performed as indicated . extracellular acidification rates ( ECAR ) were measured with a Seahorse Extracellular Flux Analyzer XF96 . data are presented as mean ± SD ( n = 3 independent experiments ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. 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 of basal 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 .

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

D. 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 ) ATP production ( F ) were measured in control or OPA1 KO HCT116 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 .

O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, 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 were maintained in normoxia or hypoxia ( 1 % O2 ) for 24 hours . glucose uptake ( G ) lactate production ( H ) were measured , values were normalized to . 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 O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A . Control OMA1 KO HCT116 were cultured in normoxia or in hypoxia ( 1 % O2 ) for 24 or 48 hours . whole lysates were analyzed by Western blotting with antibodies against HIF - 1α , HK2 , LDHA , PKM2 , GPI , OMA1 , or β - Actin . relative were further evaluated by densitometry using ImageJ software were presented as bar graphs in 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 .

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

A. 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 were untreated ( - ) or CoCl2 - treated ( + ) for 24 hours with 10 mM MG132 or vehicle added for last 4 hours . lysates were assessed by Western blotting with antibodies against HIF - 1α or β - Actin . Relative were further evaluated by densitometry . Error bars indicate mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) of optimum cutting ( OCT ) covered tissues from colons of WT 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 . of DHE . 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 .

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

C. 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 cultured in normoxia or hypoxia ( 1 % O2 ) for 12 hours were stained with mitoSOX , then analyzed by confocal microscopy ( representative data from three independent experiments ) . F . of mitoSOX was further analyzed by ImageJ software . Bars indicate mean ± SD of three independent experiments by a two - way ANOVA , * * * p < 0 . 001 .

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

E. 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 were cultured in normoxia or hypoxia for 12 hours , were treated with DMSO , NAC ( 10 mM ) or Rotenone ( 1 μM ) for 12 hours at same time . whole lysates were analyzed by Western blotting with antibodies against HIF - 1α , Tubulin ( G ) . glucose uptake ( H ) lactate production ( I ) were measured using microplate reader , values were normalized to . Error bars indicate 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 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O 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-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A . 293T were transiently co - transfected with empty vector NDUFB5 - MYC , or OMA1 - E324Q - Flag NDUFB5 - MYC . lysates were used for co - immunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with anti - Flag or anti - MYC antibodies ( n = 3 independent experiments ) .

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

A. 293T cells were transiently co-transfected with empty vector and NDUFB5-MYC, or OMA1-E324Q-Flag and NDUFB5-MYC. Cell lysates were used for co-immunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with anti-Flag or anti-MYC antibodies (n = 3 independent experiments).


B . 293T were transiently co - transfected with empty vector NDUFB6 - GFP , or OMA1 - E324Q - Flag NDUFB6 - GFP . lysates were used for co - immunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with antibodies against Flag or GFP ( n = 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 293T cells were transiently co-transfected with empty vector and NDUFB6-GFP, or OMA1-E324Q-Flag and NDUFB6-GFP. Cell lysates were used for co-immunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).


C . 293T were transiently co - transfected with empty vector COX4L1 - GFP , or OMA1 - E324Q - Flag COX4L1 - GFP . lysates were used for coimmunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with anti - Flag or anti - GFP antibodies ( n = 3 independent experiments ) .

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

C. 293T cells were transiently co-transfected with empty vector and COX4L1-GFP, or OMA1-E324Q-Flag and COX4L1-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with anti-Flag or anti-GFP antibodies (n = 3 independent experiments).


D . 293T were transiently co - transfected with empty vector NDUFA4 - GFP , or OMA1 - E324Q - Flag NDUFA4 - GFP . lysates were used for coimmunoprecipitation with anti - Flag M2 affinity gel at 4 ° C overnight , followed by Western blotting with antibodies against Flag or GFP ( n = 3 independent experiments ) .

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

D. 293T cells were transiently co-transfected with empty vector and NDUFA4-GFP, or OMA1-E324Q-Flag and NDUFA4-GFP. Cell lysates were used for coimmunoprecipitation with anti-Flag M2 affinity gel at 4°C overnight, followed by Western blotting with antibodies against Flag or GFP (n = 3 independent experiments).


E . HCT116 stably expressing control ( empty vector ) or OMA1 - Flag , HCT116 infected with control or shOMA1 ( OMA1 knockdown ) lentiviral particles further cultured for 5 days , were then treated with DMSO or 20 μm CCCP for 4 hours . lysates were analyzed by Western blotting with antibodies against NDUFA4 , COX4L1 , NDUFB5 , DNUFB6 , MT - CO2 , OMA1 or Tubulin . black arrowhead indicated full - COX4L1 or NDUFB6 , red arrowhead pointed to band . asterisk indicates a nonspecific band .

O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. HCT116 cells stably expressing control (empty vector) or OMA1-Flag, and HCT116 cells infected with control or shOMA1 (OMA1 knockdown) lentiviral particles and further cultured for 5 days, were then treated with DMSO or 20 μm CCCP for 4 hours. Cell lysates were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, MT-CO2, OMA1 or Tubulin. The black arrowhead indicated the full-length COX4L1 or NDUFB6, and the red arrowhead pointed to the cleaved band. The asterisk indicates a nonspecific band.


F . WT , OMA1 KO OMA1 KO HCT116 expressing WT - OMA1 ( OMA1 - Flag ) or proteolytic inactive OMA1 ( E324Q - Flag ) were treated with CHX ( 100 µg / ml ) plus CCCP ( 20 µM ) for 0 , 2 , 4 , 6 hours . lysates were then assessed by western blot with antibodies against NDUFA4 , COX4L1 , NDUFB5 , NDUFB6 , OMA1 or HSPD1 ( representative data from three independent experiments ) . asterisk indicates a nonspecific band .

O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were treated with CHX (100 µg/ml) plus CCCP (20 µM) for 0, 2, 4, 6 hours. The cell lysates were then assessed by western blot with antibodies against NDUFA4, COX4L1, NDUFB5, NDUFB6, OMA1 or HSPD1 (representative data from three independent experiments). The asterisk indicates a nonspecific band.


G . Control or OMA1 KO HCT116 were cultured in hypoxia ( 1 % O2 ) for 0 , 24 , or 48 hours . lysates were assessed by Western blotting with anti - NDUFB5 , anti - NDUFB6 , anti - COX4L1 , anti - NDUFA4 antibodies , or anti - β - Actin . relative were further evaluated by densitometry using ImageJ software were presented as bar graphs in 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 .

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

G. Control or OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0, 24, or 48 hours. Cell lysates were assessed by Western blotting with anti-NDUFB5, anti-NDUFB6, anti-COX4L1, anti-NDUFA4 antibodies, or anti-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.


A . Mitochondria isolated from tumors ( T ) or adjacent normal ( N ) tissues of WT OMA1 - / - mice were subjected to blue native PAGE ( BN - PAGE ) , respiratory chain complexes were analyzed by Western blotting with anti - NDUFB8 ( I ) , anti - SDHA ( II ) , anti - COX4L1 ( IV ) , anti - ATP5A1 ( V , ATP synthase ) antibodies . SDS - PAGE with antibody against HSPD1 was used as a loading control . Relative were further evaluated by densitometry using ImageJ software . 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 .

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

A. Mitochondria isolated from tumors (T) or adjacent normal (N) tissues of WT and OMA1-/- mice were subjected to blue native PAGE (BN-PAGE), and respiratory chain complexes were analyzed by Western blotting with anti-NDUFB8 (Complex I), anti-SDHA (Complex II), anti-COX4L1 (Complex IV), and anti-ATP5A1 (Complex V, ATP synthase) antibodies. SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. 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 . Digitonin - solubilized mitochondria were isolated from control OMA1 KO HCT116 cultured in normoxia or hypoxia ( 1 % O2 ) for 12 hours , then analyzed by BN - PAGE immunoblotting using indicated antibodies against NDUFB8 ( I ) , SDHA ( II ) , NDUFA4 ( IV ) , ATP5A1 ( V ) . SDS - PAGE with antibody against HSPD1 was used as a loading control . Relative were further evaluated by densitometry using ImageJ software . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . N . S . , not significant , * p < 0 . 05 .

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B. Digitonin-solubilized mitochondria were isolated from control and OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours, and then analyzed by BN-PAGE and immunoblotting using the indicated antibodies against NDUFB8 (Complex I), SDHA (Complex II), NDUFA4 (Complex IV), and ATP5A1 (Complex V). SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, *p<0.05.


C . lysates of adjacent normal tissues ( N ) or tumors ( T ) from WT OMA1 - / - mice were analyzed by Western blotting with antibodies against NDUFA4 , COX4L1 , NDUFB5 , DNUFB6 , UQCRC2 , or Tubulin . Relative were further evaluated using ImageJ software . Error bars indicate mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * * p < 0 . 001 .

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C. The lysates of adjacent normal tissues (N) or tumors (T) from WT and OMA1-/- mice were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, UQCRC2, or Tubulin. Relative protein levels were further evaluated using ImageJ software. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, ***p < 0.001.


Representative confocal of FtsZ · SlmA · SBS condensates formed in 150 g / L dextran 500 or Ficoll 70 , absence of condensates in dilute solution . Scale bars : 20 μm , except for at higher magnification in dextran ( 5 μm , second row ) .

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Representative confocal images of the FtsZ·SlmA·SBS condensates formed in 150 g/L dextran 500 or Ficoll 70, and absence of condensates in dilute solution. Scale bars: 20 μm, except for images at higher magnification in dextran (5 μm, second row).


Turbidity of FtsZ · SlmA · SBS in buffer ( n = 3 ) , in 150 g / L dextran 500 ( n = 5 ) or Ficoll 70 ( n = 3 ) in 50 g / L PEG 8 ( n = 3 ) . Data correspond to average ± S . D .

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Turbidity of FtsZ·SlmA·SBS in buffer (n = 3), in 150 g/L dextran 500 (n = 5) or Ficoll 70 (n = 3) and in 50 g/L PEG 8 (n = 3). Data correspond to the average ± S.D.


Formation of condensates as a function of FtsZ SlmA · SBS , as measured by turbidity , in working buffer ( 300 mM KCl ) . SlmA was fivefold that of SBS ( except at 0 . 5 μM SBS , where SlmA was 3 μM ) . Data are average of 2 independent measurements . Errors ( S . D . ) , symmetrical , are depicted as discs .

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Formation of condensates as a function of FtsZ and SlmA·SBS concentration, as measured by turbidity, in working buffer (300 mM KCl). SlmA concentration was fivefold that of SBS (except at 0.5 μM SBS, where SlmA concentration was 3 μM). Data are the average of 2 independent measurements. Errors (S.D.), symmetrical, are depicted as white discs.


Representative confocal of FtsZ · SlmA · SBS condensates at specified salt concentrations . concentrations of FtsZ , SlmA SBS were 12 , 5 1 µM , respectively . Scale bars : 5 μm . Data information : measurements in 150 g / L dextran 500 .

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Representative confocal images of the FtsZ·SlmA·SBS condensates at the specified salt concentrations. The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 µM, respectively. Scale bars: 5 μm. Data information: All measurements in 150 g/L dextran 500.


Representative confocal showing final state after addition of FtsZ - Alexa 488 to FtsZ · SlmA · SBS complexes ( FtsZ labeled with Alexa 647 ) in 150 g / L dextran . Below , showing stepwise diffusion of FtsZ - Alexa 488 into condensates containing FtsZ - Alexa 647 at indicated times in seconds ( time zero , beginning of visualization for that particular condensate ) corresponding profiles at selected times in green channel . profile in red channel is shown as a reference varies slightly among . Scale bars : 5 μm ( top row ) 4 μm ( bottom row ) .

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Representative confocal images showing final state after addition of FtsZ-Alexa 488 to FtsZ·SlmA·SBS complexes (FtsZ labeled with Alexa 647) in 150 g/L dextran. Below, images showing the stepwise diffusion of FtsZ-Alexa 488 into the condensates containing FtsZ-Alexa 647 at the indicated times in seconds (time zero, beginning of visualization for that particular condensate) and corresponding intensity profiles at selected times in the green channel. The profile in the red channel is shown as a reference and varies slightly among images. Scale bars: 5 μm (top row) and 4 μm (bottom row).


Stepwise diffusion of FtsZ - Alexa 488 into FtsZ · SlmA · SBS condensates ( FtsZ labeled with Alexa 647 ) at indicated times in seconds ( time zero , beginning of visualization for those particular condensates ) in 100 g / L PEG . Scale bars : 4 μm .

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Stepwise diffusion of FtsZ-Alexa 488 into FtsZ·SlmA·SBS condensates (FtsZ labeled with Alexa 647) at the indicated times in seconds (time zero, beginning of visualization for those particular condensates) in 100 g/L PEG. Scale bars: 4 μm.


Assembly of FtsZ upon GTP addition ( 0 . 5 mM ) to FtsZ · SlmA · SBS condensates condensates formed after FtsZ disassembly at indicated times in minutes ( time zero , GTP addition ) in 150 g / L dextran . Scale bars : 5 μm . Scheme of dynamic process on right . number of condensates decreases upon formation , they rearrange upon GTP depletion disassembly .

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Assembly of FtsZ fibers upon GTP addition (0.5 mM) to FtsZ·SlmA·SBS condensates and condensates formed after FtsZ fibers disassembly at the indicated times in minutes (time zero, GTP addition) in 150 g/L dextran. Scale bars: 5 μm. Scheme of the dynamic process on the right. The number of condensates decreases upon fibers formation, and they rearrange upon GTP depletion and fibers disassembly.


B - D . Representative confocal of microdroplets without ( B ) with GTP ( C D ) . concentrations of FtsZ , SlmA SBS were 12 , 5 1 μM , respectively ( B C ) or 6 , 3 0 . 5 μM respectively ( D ) . 1 mM ( C ) or 2 mM GTP ( D ) . Scale bars : 40 μm except in on far right ( 20 μm ) , which are either a magnification of indicated region in merged image ( B ) or independent at higher magnification ( C , D ) .

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B-D. Representative confocal images of the microdroplets without (B) and with GTP (C and D). The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 μM, respectively (B and C) or 6, 3 and 0.5 μM respectively (D). 1 mM (C) or 2 mM GTP (D). Scale bars: 40 μm except in images on the far right (20 μm), which are either a magnification of the indicated region in the merged image (B) or independent images at higher magnification (C, D).


B , C . Representative confocal of microdroplets stabilized by E . coli lipid mixture containing biphasic PEG / mixture FtsZ · SlmA · SBS without with 2 mM GTP , respectively . Last image in ( B ) focuses in lipid interface to show high density of condensates . Scale bars : 20 μm except in on far right ( 10 μm ) , which are an independent image at higher magnification ( B ) or a magnification of indicated region in merged image ( C ) .

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B,C. Representative confocal images of the microdroplets stabilized by the E. coli lipid mixture containing the biphasic PEG/DNA mixture and the FtsZ·SlmA·SBS complex without and with 2 mM GTP, respectively. Last image in (B) focuses in the lipid interface to show the high density of condensates. Scale bars: 20 μm except in images on the far right (10 μm), which are an independent image at higher magnification (B) or a magnification of the indicated region in the merged image (C).


( A ) A431 WT SKO - / + GFP - fused WT or A212P - seipin were delipidated , treated with OA for 1 h , fixed stained with LD540 . Maximum projections of deconvolved confocal z - stacks . Dashed lines indicate boundaries . ( B ) of LD areas of treated as in ( A ) imaged with widefield microscopy . Bars : % of LDs / in indicated category , mean ± , n = 57 - 61 / genotype , 2 experiments , p * * < 0 . 005 ( unpaired T - ) .

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(A) A431 WT and SKO cells -/+ GFP-fused WT or A212P-seipin were delipidated, treated with OA for 1 h, fixed and stained with LD540. Maximum intensity projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (B) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/cell in indicated size category, mean ± SEM, n=57-61 cells/genotype, 2 experiments, p**< 0.005 (unpaired T-test).


( C ) SKO + WT - or A212P - seipin - GFP were stained for calreticulin . Maximum projections of deconvolved confocal z - stacks .

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(C) SKO + WT- or A212P-seipin-GFP cells were stained for calreticulin. Maximum intensity projections of deconvolved confocal z-stacks.


( D ) SKO + WT - or A212P - seipin - GFP were grown in standard growth medium , fixed stained with anti - GFP antibodies . Widefield STORM .

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(D) SKO + WT- or A212P-seipin-GFP cells were grown in standard growth medium, fixed and stained with anti-GFP antibodies. Widefield and STORM images.


( E ) SKO + WT - seipin - GFP were delipidated treated with OA for 1h , fixed stained with LipidTox Red . Single focal plane of a 3D - SIM z - stack . ( F ) of ( E ) , bars : % of LDs / with indicated number of WT - seipin - GFP associated , mean ± , n = 12 ( 693 LDs ) , 2 experiments , p * * < 0 . 005 ( Tukey multiple comparisons ) .

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(E) SKO + WT-seipin-GFP cells were delipidated and treated with OA for 1h, fixed and stained with LipidTox Red. Single focal plane of a 3D-SIM z-stack. (F) Analysis of (E), bars: % of LDs/cell with the indicated number of WT-seipin-GFP punctae associated, mean ± SEM, n=12 cells (693 LDs), 2 experiments, p**< 0.005 (Tukey multiple comparisons test).


( G ) SKO + WT - or A212P - seipin - GFP were delipidated , incubated with OA for 30 min , immunolabelled for with anti - GFP antibodies . Black arrowheads : immuno - gold , orange arrowheads : ER - LD junctions . See also Appendix Fig S3 . ( H ) of ( G ) , bars : relative labeling index in different compartments , n = 3456 - 4512 immuno - gold clusters . Distribution of both WT - A212P - GFP - seipin immunolabel is non - random ( chi square , * * p < 0 . 005 ) , but there is significant enrichment of WT - but not A212P - seipin - GFP at ER - LD contacts relative to ER ( binomial , * * p < 0 . 005 ) . See also Appendix Table S1 S2 .

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(G) SKO + WT- or A212P-seipin-GFP cells were delipidated, incubated with OA for 30 min, and immunolabelled for TEM with anti-GFP antibodies. Black arrowheads: immuno-gold, orange arrowheads: ER-LD junctions. See also Appendix Fig S3. (H) Analysis of (G), bars: relative labeling index in different cellular compartments, n=3456-4512 immuno-gold clusters. Distribution of both WT- and A212P-GFP-seipin immunolabel is non-random (chi square test, **p<0.005), but there is significant enrichment of WT- but not A212P-seipin-GFP at ER-LD contacts relative to the ER (binomial test, **p<0.005). See also Appendix Table S1 and S2.


( I ) A431 WT with seipin tagged with sf - GFP at its chromosomal locus stably expressing ER marker plasmid ( BFP - KDEL ) were delipidated treated with OA for 1 h , fixed stained with LipidTox Red . Single confocal plane of an airyscan z - stack .

(I) A431 WT cells with seipin tagged with sf-GFP at its chromosomal locus and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 1 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack.


( A ) SKO stably expressing WT - seipin - GFP were delipidated then incubated with OA LipidTox Deep Red for 30 - 75 min . Airyscan video snapshots , upper panel : an example of 2 seipin - GPF , one stably associated with a LD ( blue arrowhead ) another not LD associated ( orange arrowhead ) . Lower panel : an example of a mobile LD , with a single seipin moving together with it ( blue arrowhead ) . ( B ) of ( A ) , bars : mean velocity of tracked seipin ± , n = 13 ROIs from 13 , 90 , 2 experiments , * * p < 0 . 005 ( unpaired T - ) .

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(A) SKO cells stably expressing WT-seipin-GFP cells were delipidated and then incubated with OA and LipidTox Deep Red for 30-75 min. Airyscan video snapshots, upper panel: an example of 2 seipin-GPF punctae, one stably associated with a LD (blue arrowhead) and another not LD associated (orange arrowhead). Lower panel: an example of a mobile LD, with a single seipin puncta moving together with it (blue arrowhead). (B) Analysis of (A), bars: mean velocity of tracked seipin puncta ± SEM, n=13 ROIs from 13 cells, 90 puncta, 2 experiments, **p<0.005 (unpaired T-test).


( C ) of LD mobility in WT SKO . were incubated with OA for 45 - 75 min , LDs were visualized with LD540 imaged by widefield microscopy . Bars : 1 - [ Pearson ' s colocalization coefficient between subsequent frames / ] , ± , n = 149 - 151 , 2 experiments , * * p < 0 . 005 ( Mann Whitney ) . ( D ) Examples of LD trajectories from treated imaged , arrowhead indicates tracked LD . ( E ) of LD mobility from ( C ) ( D ) , LDs plotted for volume mean velocity .

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(C) Analysis of LD mobility in WT and SKO cells. Cells were incubated with OA for 45-75 min, LDs were visualized with LD540 and cells imaged by widefield microscopy. Bars: 1 - [Pearson's colocalization coefficient between subsequent frames/cell], ± SEM, n= 149-151 cells, 2 experiments, **p<0.005 (Mann Whitney test). (D) Examples of LD trajectories from cells treated and imaged , arrowhead indicates the tracked LD. (E) Analysis of the LD mobility from (C) and (D), individual LDs plotted for volume and mean velocity.