( E ) Brain H & E histology at 3 dpi . The top right and both bottom panels show examples , in two different mice , of leukocyte clusters ( arrows ) alongside the ventricular wall . No such clusters were detected in the LV : : S i . m . - i . n . vaccinated mice ( top left panel ) . Scale bar : 200 μm . The close up view ( bottom right panel ) highlights the thickened , disorganized ependymal lining , compared to the normal ependymal cells and cilia of an LV : : S i . m . - i . n . vaccinated mouse ( top left panel ) . Scale bar : 50 μm .

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(E) Brain H&E histology at 3 dpi. The top right and both bottom panels show examples, in two different mice, of leukocyte clusters (arrows) alongside the ventricular wall. No such clusters were detected in the LV::S i.m.-i.n. vaccinated mice (top left panel). Scale bar: 200 μm. The close up view (bottom right panel) highlights the thickened, disorganized ependymal lining, compared to the normal ependymal cells and cilia of an LV::S i.m.-i.n. vaccinated mouse (top left panel). Scale bar: 50 μm.


( B ) Brain or lung viral RNA contents , determined by conventional E - specific or sub - genomic Esg - specific qRT - PCR at 3 dpi . Two mice out of the 5 sham - vaccinated mice did not have detectable viral RNA in the lungs despite high viral RNA content in the brain and hACE2 mRNA expression levels comparable to that of the other mice in the same group . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 ) .

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(B) Brain or lung viral RNA contents, determined by conventional E-specific or sub-genomic Esg-specific qRT-PCR at 3 dpi. Two mice out of the 5 sham-vaccinated mice did not have detectable viral RNA in the lungs despite high viral RNA content in the brain and hACE2 mRNA expression levels comparable to that of the other mice in the same group. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001).


( E ) Wild type or µMT KO mice ( n = 5 - 9 / group ) were injected by LV : : S or sham following the time line shown in ( A ) , then pretreated with Ad5 : : hACE2 4 days before challenge with the ancestral SARS - CoV - 2 strain . Lung viral RNA contents were determined at 3 dpi . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 ) .

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(E) Wild type or µMT KO mice (n = 5-9/group) were injected by LV::S or sham following the time line shown in (A), then pretreated with Ad5::hACE2 4 days before challenge with the ancestral SARS-CoV-2 strain. Lung viral RNA contents were determined at 3 dpi. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001).


D Changes ( Δ g ) in inguinal WAT ( iWAT ) mass of mice in ( C ) . n = 5 . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test . N . S . , not significant . E Changes ( Δ g ) in epididymal WAT ( eWAT ) mass of mice in ( C ) . n = 5 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 relative to GFP by Student ' s t - test . N . S . , not significant .

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D Changes (Δ g) in inguinal WAT (iWAT) mass of mice in (C). n=5. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant. E Changes (Δ g) in epididymal WAT (eWAT) mass of mice in (C). n=5. *P<0.05, **P<0.01, ***P<0.001 relative to GFP by Student's t-test. N.S., not significant.


F Expression of Pltp mRNA in indicated tissues . Data are shown by relative Pltp mRNA levels normalized by 18s expression . n = 3 for all tissues ( except BAT and WAT n = 5 , Testis n = 2 ) . * P < 0 . 05 Pltp expression in BAT vs other tissues ( except testis and lung ) by Student ' s t - test .

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F Expression of Pltp mRNA in indicated tissues. Data are shown by relative Pltp mRNA levels normalized by 18s expression. n=3 for all tissues (except BAT and WAT n=5, Testis n=2). *P<0.05 Pltp expression in BAT vs other tissues (except testis and lung) by Student's t-test.


G Plasma PLTP activity from control ( Ppargflox / flox ) and BAT - less mice ( Pparg UCP1 - KO , Ucp1 - Cre x Ppargflox / flox ) . Following 6 - h fasting plasma was obtained from PpargUCP1 - KO mice and littermate control . n = 3 for both groups . * P < 0 . 05 relative to control mice by Student ' s t - test .

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G Plasma PLTP activity from control (Ppargflox/flox) and BAT-less mice (Pparg UCP1-KO, Ucp1-Cre x Ppargflox/flox). Following 6-h fasting plasma was obtained from PpargUCP1-KO mice and littermate control. n=3 for both groups. *P < 0.05 relative to control mice by Student's t-test.


H PLTP protein levels in the culture conditioned medium from primary differentiated inguinal adipocytes of wild type ( WT ) and aP2 - PRDM16 mice [ 24 ] . Peptide levels was determined by quantitative proteomics . n = 2 for both groups .

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H PLTP protein levels in the culture conditioned medium from primary differentiated inguinal adipocytes of wild type (WT) and aP2-PRDM16 mice [24]. Peptide levels was determined by quantitative proteomics. n=2 for both groups.


I Transcriptional regulation of the Pltp gene by PRDM16 and PPARγ . ChIP - seq data for PRDM16 and PPARγ were obtained from [ 31 , 32 ] , respectively .

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I Transcriptional regulation of the Pltp gene by PRDM16 and PPARγ. ChIP-seq data for PRDM16 and PPARγ were obtained from [31,32], respectively.


B Changes in body weight of mice in ( A ) during 10 weeks of HFD . n = 9 for GFP and n = 8 for PLTP ( cohort 1 ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test .

B Changes in body weight of mice in (A) during 10 weeks of HFD. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05, **P<0.01, ***P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.


C Changes in food intake of mice in ( A ) during 10 weeks of HFD ( cohort 1 ) . n = 9 for GFP ( cage 1 = 5 mice ; cage 2 = 4 mice ) and n = 8 for PLTP ( cage 1 = 4 mice ; cage 2 = 4 mice )

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C Changes in food intake of mice in (A) during 10 weeks of HFD (cohort 1). n=9 for GFP (cage 1 = 5 mice; cage 2 = 4 mice) and n=8 for PLTP (cage 1 = 4 mice; cage 2 = 4 mice)


D Body composition of mice in ( A ) at 10 weeks of HFD determined by EchoMRI . n = 9 for GFP and n = 8 for PLTP ( cohort 1 ) . * P < 0 . 05 relative to GFP by two - way ANOVA followed by Tukey ' s test . N . S . , not significant .

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D Body composition of mice in (A) at 10 weeks of HFD determined by EchoMRI. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05 relative to GFP by two-way ANOVA followed by Tukey's test. N.S., not significant.


E Whole - body energy expenditure of mice at 3 weeks of HFD . Area under the curve ( AUC ) of VO2 is shown in the right graph . n = 7 for GFP and n = 8 for PLTP ( cohort 2 ) . * P < 0 . 05 relative to GFP by Student ' s t - test .

E Whole-body energy expenditure of mice at 3 weeks of HFD. Area under the curve (AUC) of VO2 is shown in the right graph. n=7 for GFP and n=8 for PLTP (cohort 2). *P<0.05 relative to GFP by Student's t-test.


F Physical movement of mice at 3 weeks of HFD as determined by metabolic cage analysis . n = 7 for GFP and n = 8 for PLTP ( cohort 2 ) . N . S . , not significant by Student ' s t -

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F Physical movement of mice at 3 weeks of HFD as determined by metabolic cage analysis. n=7 for GFP and n=8 for PLTP (cohort 2). N.S., not significant by Student's t-test


G OCR in the interscapular BAT and iWAT of control and PLTP - treated mice . One week after the treatment , BAT and iWAT were isolated from mice ( 7 weeks old ) that received adenovirus for GFP or PLTP . n = 5 for both groups . * P < 0 . 05 relative to GFP by Student ' s t - test . N . S . , not significant .

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G OCR in the interscapular BAT and iWAT of control and PLTP-treated mice. One week after the treatment, BAT and iWAT were isolated from mice (7 weeks old) that received adenovirus for GFP or PLTP. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.


H Gene expression of indicated thermogenic genes in ( A ) from interscapular BAT for GFP ( n = 8 ) and PLTP ( n = 8 ) groups ( cohort 1 ) except n = 7 for Ucp1 of the PLTP group . Tbp was used as an internal control . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test . N . S . , not significant .

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H Gene expression of indicated thermogenic genes in (A) from interscapular BAT for GFP (n=8) and PLTP (n=8) groups (cohort 1) except n=7 for Ucp1 of the PLTP group. Tbp was used as an internal control. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant.


I H & E staining of interscapular BAT from mice in ( A ) at 10 weeks of HFD ( cohort 1 ) . Representative images from n = 3 mice for both groups . Scale Bar , 50µm .

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I H&E staining of interscapular BAT from mice in (A) at 10 weeks of HFD (cohort 1). Representative images from n=3 mice for both groups. Scale Bar, 50µm.


A Glucose tolerance test of mice that received AAV control ( GFP ) or PLTP at 10 weeks of HFD . After eight hours of fasting , 2 g / kg body weight of glucose was administered . n = 9 for GFP and n = 7 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test .

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A Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 10 weeks of HFD. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=9 for GFP and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.


B Insulin tolerance test of mice in ( A ) at 10 weeks of HFD . Mice were fasted for four hours and administered insulin ( 0 . 75 U / kg body weight ) . n = 9 for GFP control and n = 7 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test .

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B Insulin tolerance test of mice in (A) at 10 weeks of HFD. Mice were fasted for four hours and administered insulin (0.75 U/kg body weight). n=9 for GFP control and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.


C Fasting insulin levels of mice in ( A ) at 10 weeks of HFD . Blood samples were harvested after eight hours of fasting . n = 5 for GFP and n = 4 for PLTP . * P < 0 . 05 relative to GFP by Student ' s t - test .

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C Fasting insulin levels of mice in (A) at 10 weeks of HFD. Blood samples were harvested after eight hours of fasting. n=5 for GFP and n=4 for PLTP. *P<0.05 relative to GFP by Student's t-test.


D Glucose tolerance test of mice that received AAV control ( GFP ) or PLTP at 6 weeks of HFD . Note that no difference was found in body weight between the groups by two - way ANOVA repeated measures followed by Bonferroni ' s test . . After eight hours of fasting , 2 g / kg body weight of glucose was administered . n = 7 for GFP control and n = 10 for PLTP . AUC of glucose is shown in the right graph . * P < 0 . 05 by Student ' s t - test .

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D Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 6 weeks of HFD. Note that no difference was found in body weight between the groups by two-way ANOVA repeated measures followed by Bonferroni's test.. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=7 for GFP control and n=10 for PLTP. AUC of glucose is shown in the right graph. *P<0.05 by Student's t-test.


E 18F - FDG PET / CT scan images of mice that received adenovirus GFP ( control ) or PLTP at 14 weeks of HFD . Mice received CL316 , 243 at dose of 1 mg / kg of body weight 30 min before the PET / CT images . Representative images of n = 5 for both groups . Yellow arrows indicate anatomical references as bladder , heart and interscapular BAT .

E 18F-FDG PET/CT scan images of mice that received adenovirus GFP (control) or PLTP at 14 weeks of HFD. Mice received CL316,243 at dose of 1 mg/kg of body weight 30 min before the PET/CT images. Representative images of n=5 for both groups. Yellow arrows indicate anatomical references as bladder, heart and interscapular BAT.


F Quantification of 18F - FDG uptake in indicated tissues from mice in ( E ) . After the 18F - FDG PET / CT imaging , indicated tissues were harvested and the radioactivity was measured by scintillation counter . n = 5 for both groups . * P < 0 . 05 relative to GFP by Student ' s t - test . N . S . , not significant .

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F Quantification of 18F-FDG uptake in indicated tissues from mice in (E). After the 18F-FDG PET/CT imaging, indicated tissues were harvested and the radioactivity was measured by scintillation counter. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.


A Lipidomics - based quantification of indicated lipid species in mice that received tail - vein injection of adenovirus expressing PLTP or GFP ( control ) . Blood and tissues were harvested after 10 weeks of HFD . n = 4 for GFP and n = 5 for PLTP in mice on HFD . LPC = ( lyso ) phosphatidylcholine , LPE = ( lyso ) phosphatidylethalonamine , PA = phosphatidic acid , SM = sphingomyelin , TAG = triglycerides , FFA = free fatty acid . Statistical analysis is demonstrated ( P value ) relative to GFP by Student ' s t - test .

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A Lipidomics-based quantification of indicated lipid species in mice that received tail-vein injection of adenovirus expressing PLTP or GFP (control). Blood and tissues were harvested after 10 weeks of HFD. n=4 for GFP and n=5 for PLTP in mice on HFD. LPC = (lyso)phosphatidylcholine, LPE = (lyso)phosphatidylethalonamine, PA = phosphatidic acid, SM = sphingomyelin, TAG = triglycerides, FFA = free fatty acid. Statistical analysis is demonstrated (P value) relative to GFP by Student's t-test.


B Plasma total cholesterol levels in mice that received adenovirus GFP ( control ) or PLTP . Blood and tissues were harvested seven days after adenovirus infection . n = 8 in mice on RD and n = 7 in mice on HFD for 14 weeks . * * * P < 0 . 001 relative to GFP by Student ' s t - test .

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B Plasma total cholesterol levels in mice that received adenovirus GFP (control) or PLTP. Blood and tissues were harvested seven days after adenovirus infection. n=8 in mice on RD and n=7 in mice on HFD for 14 weeks. ***P<0.001 relative to GFP by Student's t-test.


C Relative mRNA levels of indicated genes in the liver of mice on HFD . Tbp was used as an internal control . n = 4 for GFP and n = 6 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test .

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C Relative mRNA levels of indicated genes in the liver of mice on HFD. Tbp was used as an internal control. n=4 for GFP and n=6 for PLTP. *P<0.05, **P<0.01 relative to GFP by Student's t-test.


D Bile acid levels in the liver of mice in ( B ) . n = 8 in mice on RD and n = 6 in mice on HFD . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test .

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D Bile acid levels in the liver of mice in (B). n=8 in mice on RD and n=6 in mice on HFD. *P<0.05, **P<0.01 relative to GFP by Student's t-test.


E Bile acid levels in the plasma of mice in ( B ) . n = 8 in mice on RD and n = 7 in mice on HFD . * * * P < 0 . 001 relative to GFP by Student ' s t - test . F Bile acid levels in the feces of mice in ( B ) . Feces were collected from mice on RD and HFD after seven days of treatment . n = 7 in mice on RD and HFD . * * P < 0 . 01 , * * * P < 0 . 001 relative to GFP by Student ' s t - test .

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E Bile acid levels in the plasma of mice in (B). n=8 in mice on RD and n=7 in mice on HFD. ***P<0.001 relative to GFP by Student's t-test. F Bile acid levels in the feces of mice in (B). Feces were collected from mice on RD and HFD after seven days of treatment. n=7 in mice on RD and HFD. **P<0.01, ***P<0.001 relative to GFP by Student's t-test.


H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid ( CA ) . Cells were treated with CA at indicated concentrations for 24 h . 0 . 1 % ETOH was used as vehicle control ( Ctr ) . n = 3 . Ucp1 mRNA expression was normalized by Tbp as an internal control . * * P < 0 . 01 relative to Ctr by one - way ANOVA followed by Tukey ' s test . N . S . , not significant .

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H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid (CA). Cells were treated with CA at indicated concentrations for 24 h. 0.1% ETOH was used as vehicle control (Ctr). n=3. Ucp1 mRNA expression was normalized by Tbp as an internal control. **P<0.01 relative to Ctr by one-way ANOVA followed by Tukey's test. N.S., not significant.


B Relative expression of Pltp in liver of mice that received adenovirus GFP ( control ) or PLTP . n = 4 for GFP , n = 6 for PLTP and n = 6 for PLTPM159E . * * P < 0 . 01 relative to GFP by ANOVA one - way followed by Tukey ' s test . N . S . , not significant .

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B Relative expression of Pltp in liver of mice that received adenovirus GFP (control) or PLTP. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. **P<0.01 relative to GFP by ANOVA one-way followed by Tukey's test. N.S., not significant.


C Plasma PLTP concentration in mice that received adenovirus GFP or PLTP as determined by quantitative mass spectrometry . n = 4 for GFP , n = 6 for PLTP and n = 6 for PLTPM159E . * * * P < 0 . 001 relative to GFP by ANOVA one - way followed by Tukey ' s test . N . S . , not significant .

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C Plasma PLTP concentration in mice that received adenovirus GFP or PLTP as determined by quantitative mass spectrometry. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. ***P<0.001 relative to GFP by ANOVA one-way followed by Tukey's test. N.S., not significant.


D Plasma PLTP activity in mice that received adenovirus GFP or PLTP . n = 4 for GFP , n = 6 for PLTP and n = 6 for PLTPM159E . * * * P < 0 . 001 PLTP relative to GFP and PLTPM159E ; * P < 0 . 05 PLTPM159E relative to GFP by ANOVA one - way followed by Tukey ' s test .

D Plasma PLTP activity in mice that received adenovirus GFP or PLTP. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. ***P<0.001 PLTP relative to GFP and PLTPM159E; *P<0.05 PLTPM159E relative to GFP by ANOVA one-way followed by Tukey's test.


E Changes in body weight of mice that received AAV control or PLTP during 10 weeks of HFD . n = 8 for GFP , n = 10 for PLTP and n = 10 for PLTPM159E . * P < 0 . 05 , * * P < 0 . 01 relative to PLTPM159E by two - way ANOVA repeated measures followed by Bonferroni ' s test .

E Changes in body weight of mice that received AAV control or PLTP during 10 weeks of HFD. n=8 for GFP, n=10 for PLTP and n=10 for PLTPM159E. *P<0.05, **P<0.01 relative to PLTPM159E by two-way ANOVA repeated measures followed by Bonferroni's test.


F Relative Ucp1 expression in BAT of mice that received AAV GFP ( control ) or PLTP at 10 weeks of HFD . n = 4 for GFP , n = 5 for PLTP and n = 4 for PLTPM159E . Tbp was used as an internal control . * P < 0 . 05 , * * P < 0 . 01 by ANOVA one - way followed by Tukey ' s test .

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F Relative Ucp1 expression in BAT of mice that received AAV GFP (control) or PLTP at 10 weeks of HFD. n=4 for GFP, n=5 for PLTP and n=4 for PLTPM159E. Tbp was used as an internal control. *P<0.05, **P<0.01 by ANOVA one-way followed by Tukey's test.


G Glucose tolerance test of mice that received AAV GFP or PLTP at 10 weeks of HFD . n = 5 for GFP and n = 6 for both PLTP and PLTPM159E . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 PLTP relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test . AUC of glucose , shown in the right graph , was analyzed by one - way ANOVA followed by Tukey ' s test . N . S . , not significant .

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G Glucose tolerance test of mice that received AAV GFP or PLTP at 10 weeks of HFD. n=5 for GFP and n=6 for both PLTP and PLTPM159E. *P<0.05, **P<0.01, ***P<0.001 PLTP relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test. AUC of glucose, shown in the right graph, was analyzed by one-way ANOVA followed by Tukey's test. N.S., not significant.


H Plasma total cholesterol of mice that received AAV control or PLTP at 10 weeks of HFD . n = 7 for all groups . * P = 0 . 05 by ANOVA one - way followed by Fisher ' s LSD test . N . S . , not significant .

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H Plasma total cholesterol of mice that received AAV control or PLTP at 10 weeks of HFD. n=7 for all groups. *P=0.05 by ANOVA one-way followed by Fisher's LSD test. N.S., not significant.


I Plasma bile acids in ( H ) . n = 7 for all groups . * * * P < 0 . 001 PLTP relative to GFP and PLTPM159E by ANOVA one - way followed by Tukey ' s test .

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I Plasma bile acids in (H). n=7 for all groups. ***P<0.001 PLTP relative to GFP and PLTPM159E by ANOVA one-way followed by Tukey's test.


J Plasma phospholipid in ( H ) . n = 7 for all groups . * * * P < 0 . 001 PLTP and PLTPM159E relative to GFP ; * P < 0 . 05 PLTP relative to PLTPM159E by ANOVA one - way followed by Fisher ' s LSD test .

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J Plasma phospholipid in (H). n=7 for all groups. ***P<0.001 PLTP and PLTPM159E relative to GFP; *P<0.05 PLTP relative to PLTPM159E by ANOVA one-way followed by Fisher's LSD test.


Testicular maturation arrest in the patient ( WHT3759 ) . Testicular tissue obtained by biopsy was sectioned and stained with hematoxylin / eosin . Seminiferous tubules contained meiotic germ cells such as zygotene ( ZS , arrowheads ) and pachytene spermatocytes ( PS , arrows ) , but no post - meiotic germ cells such as round spermatids were detectable . Scale bar , 25 μm .

Testicular maturation arrest in the patient (WHT3759). Testicular tissue obtained by biopsy was sectioned and stained with hematoxylin/eosin. Seminiferous tubules contained meiotic germ cells such as zygotene (ZS, arrowheads) and pachytene spermatocytes (PS, arrows), but no post-meiotic germ cells such as round spermatids were detectable. Scale bar, 25 μm.


Western blot analysis of TEX11 in the testes of 3 - month - old mice with different Tex11 gene dosages . ACTB serves as a loading control .

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Western blot analysis of TEX11 in the testes of 3-month-old mice with different Tex11 gene dosages. ACTB serves as a loading control.


Testis weight of 3 - month - old males .

Testis weight of 3-month-old males.


Sperm count of 3-month-old males.


Histological analysis of testes from adult wild - type , Tex11 KI / KO , and Tex11 − / Y males . RS , round spermatids ; ES , elongating spermatids ; Pa , pachytene spermatocytes . Scale bar , 50 μm .

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Histological analysis of testes from adult wild-type, Tex11 KI/KO, and Tex11−/Y males. RS, round spermatids; ES, elongating spermatids; Pa, pachytene spermatocytes. Scale bar, 50 μm.


The Tex11 knockin allele rescues chromosomal synapsis defects in Tex11 − / Y spermatocytes in 3 - month - old males . Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male ; for each genotype , three males were analyzed . Abbreviations for Tex11 genotypes : − / Y , Tex11 knockout ( Yang et al , 2008 ) ; KI ; − / Y , Tex11 knockin and knockout ; + / Y , wild type ; KI ; + / Y , Tex11 knockin plus wild - type Tex11 .

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The Tex11 knockin allele rescues chromosomal synapsis defects in Tex11−/Y spermatocytes in 3-month-old males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed. Abbreviations for Tex11 genotypes: −/Y, Tex11 knockout (Yang et al, 2008); KI;−/Y, Tex11 knockin and knockout; +/Y, wild type; KI;+/Y, Tex11 knockin plus wild-type Tex11.


Reduced testis weight in juvenile but not adult Tex11 KI ; − / Y males . The time frames of the first wave and adult waves of spermatogenesis are indicated .

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Reduced testis weight in juvenile but not adult Tex11 KI; −/Y males. The time frames of the first wave and adult waves of spermatogenesis are indicated.


Increased chromosomal asynapsis in spermatocytes and reduced levels of TEX11 protein in the testes of 25 - day - old Tex11 KI / KO males . Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male ; for each genotype , three males were analyzed .

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Increased chromosomal asynapsis in spermatocytes and reduced levels of TEX11 protein in the testes of 25-day-old Tex11 KI/KO males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed.


Pachytene spermatocytes from 25 - day - old Tex11 KI / KO males contain significantly fewer MLH1 foci compared to wild - type spermatocytes . Values are shown as average ± standard deviation . Error bar is standard deviation .

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Pachytene spermatocytes from 25-day-old Tex11 KI/KO males contain significantly fewer MLH1 foci compared to wild-type spermatocytes. Values are shown as average ± standard deviation. Error bar is standard deviation.


The number of MLH1 foci in pachytene spermatocytes from 3 - month - old males positively correlates with increasing Tex11 gene dosage .

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The number of MLH1 foci in pachytene spermatocytes from 3-month-old males positively correlates with increasing Tex11 gene dosage.


Quantification of MLH1 foci in pachytene oocytes from the ovaries of Tex11 − / − , Tex11 + / − , and wild - type embryonic day - 17 . 5 ( E17 . 5 ) fetuses reveals a positive correlation of Tex11 gene dosage and the number of MLH1 foci .

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Quantification of MLH1 foci in pachytene oocytes from the ovaries of Tex11−/−, Tex11+/−, and wild-type embryonic day-17.5 (E17.5) fetuses reveals a positive correlation of Tex11 gene dosage and the number of MLH1 foci.


A Western blot analysis reveals comparable levels of TEX11 protein in the testes of wild - type and point mutant KI / KO males . ACTB serves as a control .

A Western blot analysis reveals comparable levels of TEX11 protein in the testes of wild-type and point mutant KI/KO males. ACTB serves as a control.


B - D Body weight ( B ) , testisweight ( C ) in Tex11 KI ( V749A ) / KO males .

B-D Body weight (B), testisweight (C) in Tex11 KI(V749A)/KO males.


B - D Significantly reduced sperm count ( per pair of epididymides ) in Tex11 KI ( V749A ) / KO males ( D ) .

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B-D Significantly reduced sperm count (per pair of epididymides) in Tex11 KI(V749A)/KO males (D).


E Mating test of 3 - to 5 - month - old males .

E Mating test of 3- to 5-month-old males.


F Dramatically increased chromosomal asynapsis in spermatocytes from Tex11 KI ( V749A ) / KO males . 100 pachytene spermatocytes per mouse were examined by surface spread analysis .

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F Dramatically increased chromosomal asynapsis in spermatocytes from Tex11 KI(V749A)/KO males. 100 pachytene spermatocytes per mouse were examined by surface spread analysis.


G Number of MLH1 foci in pachytene spermatocytes . Note a significantly higher number of MLH1 foci in spermatocytes from KI ( Q173R ) / KO males .

G Number of MLH1 foci in pachytene spermatocytes. Note a significantly higher number of MLH1 foci in spermatocytes from KI(Q173R)/KO males.


Reverse Transcriptase PCR showing that bd1971 , and control gene dnaK , are expressed throughout the predatory cycle of Bdellovibrio bacteriovorus . RNA was isolated at the time points indicated across the top of the gel during one round of synchronous Bdellovibrio infection of E . coli cells . Primers were designed to anneal specifically to the gene of interest . L = 100bp DNA ladder , AP = Attack Phase cells , 15 - 45 = 15 - 45 minutes respectively since infection , 1h - 4h = hours respectively since infection . Ec = E . coli strain S17 - 1 RNA ( negative control : no Bdellovibrio RNA ) ; NT = control with no template RNA ; gen = B . bacteriovorus HD100 genomic DNA ( positive control ) . The cartoon above represents each stage in the predatory cycle .

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Reverse Transcriptase PCR showing that bd1971, and control gene dnaK, are expressed throughout the predatory cycle of Bdellovibrio bacteriovorus. RNA was isolated at the time points indicated across the top of the gel during one round of synchronous Bdellovibrio infection of E. coli cells. Primers were designed to anneal specifically to the gene of interest. L = 100bp DNA ladder, AP = Attack Phase cells, 15-45 = 15-45 minutes respectively since infection, 1h-4h = hours respectively since infection. Ec = E. coli strain S17-1 RNA (negative control: no Bdellovibrio RNA); NT = control with no template RNA; gen = B. bacteriovorus HD100 genomic DNA (positive control). The cartoon above represents each stage in the predatory cycle.


Epifluorescent images of E . coli invaded by B . bacteriovorus HD100 with mCherry tagged Bd1971 at the times indicated throughout the invasive process during one round of synchronous infection . Fluorescence was acquired with a two second exposure and maximum sensitivity gain but different balance and contrast levels in the fluorescence channel were used to produce these images ( see methods ) as the fluorescence was fainter at later timepoints but we wished to show whether or not the cellular localization within the Bdellovibrio altered during predation . Fluorescence images and merges with phase contrast images are shown . Scale bars are 2 µm . Images are representative of three independent experiments .

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Epifluorescent images of E. coli invaded by B. bacteriovorus HD100 with mCherry tagged Bd1971 at the times indicated throughout the invasive process during one round of synchronous infection. Fluorescence was acquired with a two second exposure and maximum sensitivity gain but different balance and contrast levels in the fluorescence channel were used to produce these images (see methods) as the fluorescence was fainter at later timepoints but we wished to show whether or not the cellular localization within the Bdellovibrio altered during predation. Fluorescence images and merges with phase contrast images are shown. Scale bars are 2 µm. Images are representative of three independent experiments.


Relative levels of c - di - GMP in matched cell biomass host - independent ( HI ) cultures of bd1971 : : kan compared with fliC1 merodiploid KnR HI control strain ( Wild - Type for bd1971 ) . The bd1971 mutant strain contains significantly more c - di - GMP than the control . Data are from 3 independent experiments .

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Relative levels of c-di-GMP in matched cell biomass host-independent (HI) cultures of bd1971::kan compared with fliC1 merodiploid KnR HI control strain (Wild-Type for bd1971). The bd1971 mutant strain contains significantly more c-di-GMP than the control. Data are from 3 independent experiments.


Relative levels of c - di - GMP in matched cell biomass wild - type predatory cultures of HD100 strain , silent Bd1971 deletion strain ( Δ1971 ) , Bd1971D306307A strain ( bd1971DD ) and Bd1971R67D strain ( bd1971R ) . All mutant strains had higher levels of c - di - GMP compared to the wild type strain . Error bars show SEM . Data are from six independent experiments for HD100 and ΔBd1971 and from four independent experiments for Bd1971DD and Bd1971R mutants .

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

Relative levels of c-di-GMP in matched cell biomass wild-type predatory cultures of HD100 strain, silent Bd1971 deletion strain (Δ1971), Bd1971D306307A strain (bd1971DD) and Bd1971R67D strain (bd1971R). All mutant strains had higher levels of c-di-GMP compared to the wild type strain. Error bars show SEM. Data are from six independent experiments for HD100 and ΔBd1971 and from four independent experiments for Bd1971DD and Bd1971R mutants.


Apo - Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations ( 2 . 5 mM ) of cAMP , cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP ( 5 mM ) was measured spectrophotometrically . Values are the average of three repeats , error bars represent standard deviation .

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Apo-Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations (2.5 mM) of cAMP, cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP (5 mM) was measured spectrophotometrically. Values are the average of three repeats, error bars represent standard deviation.


HPLC traces of reaction products generated from 100 µM c - di - GMP incubated with apo - Bd1971 ( Control ) , or protein supplemented with either 100 µM cAMP or cGMP . Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure .

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HPLC traces of reaction products generated from 100 µM c-di-GMP incubated with apo-Bd1971 (Control), or protein supplemented with either 100 µM cAMP or cGMP. Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure.


ITC titration curves and relative heats of binding for ( A ) cAMP binding to Bd1971ΔEAL . Successive injections of 6 µl of cNMP ( 0 . 5 - 1 . 6 mM ) were made into 1 . 4 ml of apo - Bd1971ΔEAL ( 50 - 150 µM ) and heat changes measured . The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment .

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ITC titration curves and relative heats of binding for (A) cAMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.


ITC titration curves and relative heats of binding for cGMP binding to Bd1971ΔEAL . Successive injections of 6 µl of cNMP ( 0 . 5 - 1 . 6 mM ) were made into 1 . 4 ml of apo - Bd1971ΔEAL ( 50 - 150 µM ) and heat changes measured . The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment .

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ITC titration curves and relative heats of binding for cGMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.


Data were fitted with an allosteric sigmoidal model in GraphPad Prism software and used to determine the kinetic parameters associated with cNMP dependent phosphodiesterase activity . Data points for both sets of results are the average of three experimental replicates with the standard deviation shown by intersecting bars . Results are representative of at least three independent experiments .

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Data were fitted with an allosteric sigmoidal model in GraphPad Prism software and used to determine the kinetic parameters associated with cNMP dependent phosphodiesterase activity.Data points for both sets of results are the average of three experimental replicates with the standard deviation shown by intersecting bars. Results are representative of at least three independent experiments.


Spot tests of co - transformants of either Bd0367 ( DgcA ; GGDEF protein ) response regulator domain or Bd3125 ( CdgA ; a degenerate GGDEF protein likely involved in c - di - GMP binding and signalling ) with full length Bd1971 , or the N - terminal ( 1 - 145 ) , or C - terminal ( 145 - 400 ) portions of Bd1971 in either pUT18c or pKT25 as indicated . Positive control was co - transformants with pUT18c - zip and pKT25 - zip , negative control was the empty vectors . The co - transformants were plated on LB - X - Gal medium with positive interaction resulting in blue colouration , which can be seen for all of the interactions in both plasmid combinations , but not for the negative control . Images are representative of at least 4 independent experiments .

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Spot tests of co-transformants of either Bd0367 (DgcA; GGDEF protein) response regulator domain or Bd3125 (CdgA; a degenerate GGDEF protein likely involved in c-di-GMP binding and signalling) with full length Bd1971, or the N-terminal (1-145), or C-terminal (145-400) portions of Bd1971 in either pUT18c or pKT25 as indicated. Positive control was co-transformants with pUT18c-zip and pKT25-zip, negative control was the empty vectors. The co-transformants were plated on LB-X-Gal medium with positive interaction resulting in blue colouration, which can be seen for all of the interactions in both plasmid combinations, but not for the negative control. Images are representative of at least 4 independent experiments.


Confirmation of BTH interactions by β - Galactosidase activity assays . The interaction is significantly stronger between Bd3125 and the N - terminal domain of Bd1971 than the interaction with the C - terminus of Bd1971 . The response regulator domain of Bd0367 showed significant interaction with Bd1971 , but the full length Bd0367 did not . * p > 0 . 05 by Student ' s t - test N / S - not significant . Data are from at least three independent experiments , error bars are the standard error of the

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Confirmation of BTH interactions by β-Galactosidase activity assays. The interaction is significantly stronger between Bd3125 and the N-terminal domain of Bd1971 than the interaction with the C-terminus of Bd1971. The response regulator domain of Bd0367 showed significant interaction with Bd1971, but the full length Bd0367 did not. *p>0.05 by Student's t-test N/S- not significant. Data are from at least three independent experiments, error bars are the standard error of the mean


A RNA - seq . of freshly isolated , proliferating and differentiating satellite cells reveals an increased expression of linc - MYH in proliferating and differentiating MuSCs , but no expression is found in in freshly isolated MuSCs .

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A RNA-seq. of freshly isolated, proliferating and differentiating satellite cells reveals an increased expression of linc-MYH in proliferating and differentiating MuSCs, but no expression is found in in freshly isolated MuSCs.


Expression of linc - MYH is confined to skeletal muscle in mouse ( B ) tissues .

Expression of linc-MYH is confined to skeletal muscle in mouse (B) tissues.


Expression of linc - MYH is confined to skeletal muscle in human ( C ) tissues .

Expression of linc-MYH is confined to skeletal muscle in human (C) tissues.


D Expression of linc - MYH is not detected in limb buds at E10 . 5 ( n = 3 ) , E13 . 5 ( n = 3 ) and E15 . 5 ( n = 3 ) . Expression of linc - MYH in hindlimb muscle of E18 . 5 , newborn and 7 day old animals ( n = 3 / 3 / 3 ) . Expression of linc - MYH in m . soleus ( soleus ) , m . tibialis anterior ( TA ) and and m . extensor digitorum longus ( EDL ) muscle at 3 weeks ( n = 3 ) and 8 weeks of age ( n = 5 ) . Biological replicates were used for all PCR experiments , data are mean ± S . E . M .

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D Expression of linc-MYH is not detected in limb buds at E10.5 (n = 3), E13.5 (n = 3) and E15.5 (n = 3). Expression of linc-MYH in hindlimb muscle of E18.5, newborn and 7 day old animals (n = 3/3/3). Expression of linc-MYH in m. soleus (soleus), m. tibialis anterior (TA) and and m. extensor digitorum longus (EDL) muscle at 3 weeks (n = 3) and 8 weeks of age (n = 5). Biological replicates were used for all PCR experiments, data are mean ± S.E.M.


E Murine linc - Myh is localized in nuclear extracts of C2C12 cells . Xist ( nuclear ) and Gapdh ( mainly cytoplasmic ) were used as controls ( n = 2 nuclear / 3 cytoplasmic biological replicates . ) Data are mean ± S . E . M .

E Murine linc-Myh is localized in nuclear extracts of C2C12 cells. Xist (nuclear) and Gapdh (mainly cytoplasmic) were used as controls (n = 2 nuclear / 3 cytoplasmic biological replicates.) Data are mean ± S.E.M.


F - H RNA - FISH identifies linc - Myh in nuclei ( DAPI , blue ) of C2C12 myoblasts . ( G ) Adipor ( cytoplasmic ) and ( H ) Xist ( nuclear ) were used as controls .

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F-H RNA-FISH identifies linc-Myh in nuclei (DAPI, blue) of C2C12 myoblasts. (G) Adipor (cytoplasmic) and (H) Xist (nuclear) were used as controls.


( J ) Heat map of proteins pulled down in the same experiments as in I , demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc - MYH . Blue color indicates pull - down of the respective protein , white color indicates that the protein was not detected in the respective sample .

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(J) Heat map of proteins pulled down in the same experiments as in I, demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc-MYH. Blue color indicates pull-down of the respective protein, white color indicates that the protein was not detected in the respective sample.


RNA - IP experiments using an anti - INO80 antibody to isolate the INO80 complex from murine C2C12 cells ( K ) INO80 in L refer to replicate INO80 precipitation experiments . Precipitated RNAs were detected by RT - PCR .

RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from murine C2C12 cells (K) INO80 in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.


RNA - IP experiments using an anti - INO80 antibody to isolate the INO80 complex from human HSMM cells ( L ) . INO80 II in L refer to replicate INO80 precipitation experiments . Precipitated RNAs were detected by RT - PCR .

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RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from human HSMM cells (L). INO80 II in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.


Body weight ( A ) of male linc - MYH KO and ctrl mice at 10 weeks ( n = 24 KO / 17 WT ; Student ' s t test , two - tailed , * * p < 0 . 01 )

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Body weight (A) of male linc-MYH KO and ctrl mice at 10 weeks (n = 24 KO/17 WT; Student's t test, two-tailed, **p < 0.01)


tibia length ( B ) of male linc - MYH KO and ctrl mice of 10 weeks ( n = 9 KO / 13 WT ; Student ' s t test , two - tailed , not significant ) . Data are mean ± S . E . M .

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tibia length (B) of male linc-MYH KO and ctrl mice of 10 weeks (n = 9 KO/13 WT; Student's t test, two-tailed, not significant). Data are mean ± S.E.M.


C , D Weight of m . tibialis anterior ( TA ) and m . digitorum longus ( EDL ) of male linc - MYH KO and ctrl mice at 10 weeks ( n = 9 KO / 10 WT for TA , n = 10 KO / 11 WT for EDL ; Student ' s t test , two - tailed , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data are mean ± S . E . M .

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C, D Weight of m. tibialis anterior (TA) and m. digitorum longus (EDL) of male linc-MYH KO and ctrl mice at 10 weeks (n = 9 KO/10 WT for TA, n = 10 KO/11 WT for EDL; Student's t test, two-tailed, **p < 0.01, ***p<0.001). Data are mean ± S.E.M.


Size distribution of fibers in cross sections of TA ( E , F ) and EDL ( G , H ) muscle in male linc - MYH KO ( blue ) and ctrl ( grey ) animals at 10 weeks . A significant increase of fiber size was observed for TA and EDL muscles of linc - MYH KO mice ( n = 5 KO / 4 WT for TA , n = 3 KO / 3 WT for EDL muscle , two - way ANOVA with Fisher ' s multiple comparisons ; * p < 0 . 05 , * * p < 0 . 01 ; > 250 fibers per animal were counted ) . Data are mean ± S . E . M .

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Size distribution of fibers in cross sections of TA (E, F) and EDL (G, H) muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.


Size distribution of fibers in cross sections of TA and EDL muscle in male linc - MYH KO ( blue ) and ctrl ( grey ) animals at 10 weeks . A significant increase of fiber size was observed for TA and EDL muscles of linc - MYH KO mice ( n = 5 KO / 4 WT for TA , n = 3 KO / 3 WT for EDL muscle , two - way ANOVA with Fisher ' s multiple comparisons ; * p < 0 . 05 , * * p < 0 . 01 ; > 250 fibers per animal were counted ) . Data are mean ± S . E . M .

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Size distribution of fibers in cross sections of TA and EDL muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.


K Numbers of myonuclei in cross sections of TA and EDL muscle ( TA : n = 6 KO / 6 WT , EDL n = 3 KO / 3 WT animals , Mann - Whitney test one - tailed , * * p < 0 . 01 , * p = 0 . 05 ) Data are mean ± S . E . M .

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K Numbers of myonuclei in cross sections of TA and EDL muscle (TA: n = 6 KO/6 WT, EDL n = 3 KO/3 WT animals, Mann-Whitney test one-tailed, **p<0.01, *p = 0.05) Data are mean ± S.E.M.


L - O Number of myonuclei / fiber and of Pax7pos ( red ) MuSCs ( red arrows ) on myofibers isolated from flexor digitorum brevis muscle . Nuclei were stained using DAPI ( blue ) . Scale bar in L indicates 50 µm for L and M . ( N ) The number of myonuclei / isolated fiber is increased in linc - MYH KO compared to WT myofibers ( n = 4 KO / 4 WT , > 26 fibers / animal ; Mann - Whitney test two - tailed , * p < 0 . 05 ) . ( O ) Number of MuSCs on isolated fibers from linc - MYH KO and WT mice ( n = 4 KO / 4 WT animals , Mann - Whitney test two - tailed ; * * * * p < 0 . 0001 ) . Data are mean ± S . E . M .

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L-O Number of myonuclei / fiber and of Pax7pos (red) MuSCs (red arrows) on myofibers isolated from flexor digitorum brevis muscle. Nuclei were stained using DAPI (blue). Scale bar in L indicates 50 µm for L and M. (N) The number of myonuclei / isolated fiber is increased in linc-MYH KO compared to WT myofibers (n = 4 KO/4 WT, > 26 fibers/animal; Mann-Whitney test two-tailed, * p < 0.05). (O) Number of MuSCs on isolated fibers from linc-MYH KO and WT mice (n = 4 KO/4 WT animals, Mann-Whitney test two-tailed; ****p < 0.0001). Data are mean ± S.E.M.


P - S Number of Pax7 ( red ) positive MuSCs in cross sections of tibialis anterior muscle ( P - R ) ; the relative amount of quiescent MuSCs identified by double staining ( yellow arrows ) for Pax7 and CalcR ( green ) does not significantly differ between WT and linc - MYH KO animals ( S ) . Nuclei were stained with DAPI ( blue ) . Scale bar in P indicates 25 µm for P and Q . ( n = 3 KO / 3 WT animals , Mann - Whitney test one - tailed ; * p = 0 . 05 , ns : not significant ) . All data are mean ± S . E . M .

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P-S Number of Pax7 (red) positive MuSCs in cross sections of tibialis anterior muscle (P-R); the relative amount of quiescent MuSCs identified by double staining (yellow arrows) for Pax7 and CalcR (green) does not significantly differ between WT and linc-MYH KO animals (S). Nuclei were stained with DAPI (blue). Scale bar in P indicates 25 µm for P and Q. (n = 3 KO/3 WT animals, Mann-Whitney test one-tailed; *p = 0.05, ns: not significant). All data are mean ± S.E.M.


T The number of MuSC - related events in FACS experiments is significantly increased in linc - MYH - KO animals ( n = 7 KO / 7 WT , Student ' s t test , two - tailed , * p = 0 . 0126 ) . Data are mean ± S . E . M .

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T The number of MuSC-related events in FACS experiments is significantly increased in linc-MYH-KO animals (n = 7 KO/7 WT, Student's t test, two-tailed, *p = 0.0126). Data are mean ± S.E.M.


U - V Structure of MuSCs and the heterochromatin content of MuSC nuclei is not changed in linc - MYH KO ( V ) compared to WT mice ( U ) .

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U-V Structure of MuSCs and the heterochromatin content of MuSC nuclei is not changed in linc-MYH KO (V) compared to WT mice (U).


RNA in situ hybridization proximity ligation assay ( rISH - PLA ) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al . . ( B - C ) rISH - PLA confirms the proximity of linc - MYH to endogenous INO80 - V5 in the nucleus of proliferating WT MuSCs ( B ) . The specific signal is lost in linc - MYH deficient MuSCs ( C ) . ( n = 3 WT / 3 KO biological replicates ; Mann - Whitney test , * p = 0 . 05 ) . Data are mean ± S . E . M .

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RNA in situ hybridization proximity ligation assay (rISH-PLA) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al.. (B-C) rISH-PLA confirms the proximity of linc-MYH to endogenous INO80-V5 in the nucleus of proliferating WT MuSCs (B). The specific signal is lost in linc-MYH deficient MuSCs (C). (n = 3 WT/3 KO biological replicates; Mann-Whitney test, *p = 0.05). Data are mean ± S.E.M.


E - I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 ( green ) and EdU incorporation ( red ) . MuSCs without ( green arrows ) and with EdU - labeling ( red arrows ) are indicated . ( E , F ) Deletion of linc - MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU - positive MuSCs . ( G - I ) The increase in both the number of MuSCs and EdU positive MuSCs in linc - MYH mutant muscle is abolished by constitutive ( G : Pax7 - Crepos / INO80 - / - , Ino80 KO ; H : linc - MYH - / - / Pax7 - Crepos / INO80 - / - , dKO ) and induced ( I : linc - MYH - / - / Pax7 - CreERT2pos / INO80 - / - , dKO CreERT ) deletion of INO80 in Pax7 expressing cells .

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E-I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 (green) and EdU incorporation (red). MuSCs without (green arrows) and with EdU-labeling (red arrows) are indicated. (E, F) Deletion of linc-MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU-positive MuSCs. (G-I) The increase in both the number of MuSCs and EdU positive MuSCs in linc-MYH mutant muscle is abolished by constitutive (G: Pax7-Crepos / INO80-/-, Ino80 KO; H: linc-MYH-/- / Pax7-Crepos / INO80-/-, dKO) and induced (I: linc-MYH-/- / Pax7-CreERT2pos / INO80-/-, dKO CreERT) deletion of INO80 in Pax7 expressing cells.


Statistical evaluation of the number of muscle stem cells observed in TA muscles ( J ) . ; ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) .

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Statistical evaluation of the number of muscle stem cells observed in TA muscles (J). ; (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).


Statistical evaluation of the ratio of EdU - incorporating MuSCs relative to all Pax7 - positive MuSCs ( K ) . ; ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) .

Statistical evaluation of the ratio of EdU-incorporating MuSCs relative to all Pax7-positive MuSCs (K). ; (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).


Statistical evaluation of the number of myonuclei in myofibers of TA muscles from different genotypes ( L ) ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) .

Statistical evaluation of the number of myonuclei in myofibers of TA muscles from different genotypes (L) (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).


A - D Isolated linc - MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates . Deletion of INO80 eliminates the difference between Pax7 - Crepos / INO80 - / - ( Ino80 KO ) and linc - MYH - / - / linc - MYH - / - / Pax7 - Crepos / INO80 - / - ( dKO ) . Images are representative images for cultures after 13 days in culture .

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A-D Isolated linc-MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates. Deletion of INO80 eliminates the difference between Pax7-Crepos / INO80-/- (Ino80 KO) and linc-MYH-/- / linc-MYH-/-/ Pax7-Crepos / INO80-/- (dKO). Images are representative images for cultures after 13 days in culture.


E Differences in proliferation rates are abolished by additional deletion of INO80 in MuSCs . Cell confluence was monitored by a live cell imaging analysis system for 13 days ( n = 7 WT / 6 linc - MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs ) .

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E Differences in proliferation rates are abolished by additional deletion of INO80 in MuSCs. Cell confluence was monitored by a live cell imaging analysis system for 13 days (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs).


F Statistical analysis of cell confluence after 13 days of muscle stem cell proliferation ( n = 7 WT / 6 linc - MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs , Student ' s t test , two - tailed ; * p < 0 . 05 , ns = not significant ) . Data are mean ± S . E . M .

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F Statistical analysis of cell confluence after 13 days of muscle stem cell proliferation (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs, Student's t test, two-tailed; *p < 0.05, ns = not significant). Data are mean ± S.E.M.


G - I Gene set enrichment analysis ( GSEA , broadinstitute . org ) of microarray data from proliferating MuSCs . Comparison of linc - MYH - / - vs . WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation ( G ) , enrichment of genes containing cis regulatory elements for YY1 ( H ) and p53 ( I ) . Enrichments are highly significant using the most conservative familywise - error rate method ( FWER ) . Enrichments were lost in linc - MYH - / - MuSCs after additional deletion of INO80 ( dKOs ) compared to Pax7 - Crepos / INO80 - / - MuSCs .

G-I Gene set enrichment analysis (GSEA, broadinstitute.org) of microarray data from proliferating MuSCs. Comparison of linc-MYH-/- vs. WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation (G), enrichment of genes containing cis regulatory elements for YY1 (H) and p53 (I). Enrichments are highly significant using the most conservative familywise-error rate method (FWER). Enrichments were lost in linc-MYH-/- MuSCs after additional deletion of INO80 (dKOs) compared to Pax7-Crepos / INO80-/- MuSCs.


Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G - I in linc - MYH - / - vs . WT MuSCs and in linc - MYH - / - / Pax7 - Crepos / INO80 - / - dKO vs . Pax7 - Crepos / INO80 - / - MuSCs . Expression of up - regulated genes in linc - MYH - / - was normalized after additional deletion of INO80 ( dKOs ) , indicating that effects of linc - MYH completely depend on INO80 ( WT n = 4 , linc - MYH - / - n = 4 , Pax7 - Crepos / INO80 - / - n = 4 , Pax7 - Cre dKO n = 3 animals used for independent isolations of MuSCs ) .

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Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/ Pax7-Crepos / INO80-/- dKO vs. Pax7-Crepos / INO80-/- MuSCs. Expression of up-regulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos / INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs).


A - D γ - H2AX staining of proliferating MuSCs from WT ( A ) , linc - MYH deficient ( B ) , Pax7 - Crepos / INO80 KO ( C ) , and linc - MYH / INO80 dKO ( D ) mice . Deletion of INO80 results in increased γ - H2AX signals while deletion of linc - MYH has no effects ( asterisk : low γ - H2AX signals , arrow : γ - H2AX - foci detected in nuclei , arrowhead : pan - nuclear γ - H2AX signal .

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A-D γ-H2AX staining of proliferating MuSCs from WT (A), linc-MYH deficient (B), Pax7-Crepos / INO80 KO (C), and linc-MYH/INO80 dKO (D) mice. Deletion of INO80 results in increased γ-H2AX signals while deletion of linc-MYH has no effects (asterisk: low γ-H2AX signals, arrow: γ-H2AX-foci detected in nuclei, arrowhead: pan-nuclear γ-H2AX signal.


E - G Statistical evaluation of the presence of γ - H2AX signals in MuSCs of different genotypes ( n = 3 / 3 / 3 / 3 animals used for independent isolations of MuSCs , two technical replicates for each animal and at least 164 cells were counted , Mann - Whitney test , * * p < 0 . 01 . Data are mean ± s . e . m . ) .

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E-G Statistical evaluation of the presence of γ-H2AX signals in MuSCs of different genotypes (n = 3/3/3/3 animals used for independent isolations of MuSCs, two technical replicates for each animal and at least 164 cells were counted, Mann-Whitney test, **p<0.01. Data are mean ± s.e.m.).


H - I Gene set enrichment analysis of microarray data obtained for proliferating MuSCs shows enrichment of genes related to activity of ATR in response to replication stress recovery ( H ) and enrichment of genes involved in chromosome maintenance ( I ) in WT compared to Pax7 - Crepos INO80 - / - MuSCs . Enrichment of these or similar gene sets was not observed when comparing WT to linc - MYH - / - MuSCs .

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H-I Gene set enrichment analysis of microarray data obtained for proliferating MuSCs shows enrichment of genes related to activity of ATR in response to replication stress recovery (H) and enrichment of genes involved in chromosome maintenance (I) in WT compared to Pax7-Crepos INO80-/- MuSCs. Enrichment of these or similar gene sets was not observed when comparing WT to linc-MYH-/- MuSCs.


A , B Co - Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc - MYH KO mice followed by Western Blot analysis . ( B ) Statistical analysis of Co - IP / Western Blot data . The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc - MYH , while the amounts of YY1 , WDR5 and TFPT pulled down by INO80 increase ( n = 3 WT / 3 linc - MYH KO animals ; one - way ANOVA , multiple comparisons by Fishers LSD test , * p < 0 . 05 ) . Data are mean ± S . E . M .

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A, B Co-Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc-MYH KO mice followed by Western Blot analysis. (B) Statistical analysis of Co-IP/Western Blot data. The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc-MYH, while the amounts of YY1, WDR5 and TFPT pulled down by INO80 increase (n = 3 WT / 3 linc-MYH KO animals; one-way ANOVA, multiple comparisons by Fishers LSD test, *p<0.05). Data are mean ± S.E.M.


C - K Proximity ligation assays between INO80 and YY1 ( C , D ) , INO80 and WDR5 ( F , G ) , and INO80 and RuvBl2 ( I , J ) in proliferating MuSCs . Red signals indicate close proximity of INO80 - V5 and the respective interacting protein . Nuclei were stained using DAPI ( blue ) . Statistical evaluation of proximity ligation assays indicating increased interactions of INO80 - V5 and YY1 ( E , n = 3 / 3 animals , 3 independent wells per animal , two - tailed students t - test , * p < 0 . 05 ) and of INO80 - V5 to WDR5 ( H , n = 2 ( WT ) / 2 ( KO ) animals , 3 independent wells per animal , two - tailed students t - test , * * p < 0 . 01 ) . No increase in proximity was detected between Ino80 - V5 and Ruvbl2 ( K , n = 2 ( WT ) / 2 ( KO ) animals , 3 independent wells per animal , two - tailed students t - test , ns : not significant ) . Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays . No signals were detected for these controls . Data are mean ± S . E . M .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K Proximity ligation assays between INO80 and YY1 (C, D), INO80 and WDR5 (F, G), and INO80 and RuvBl2 (I, J) in proliferating MuSCs. Red signals indicate close proximity of INO80-V5 and the respective interacting protein. Nuclei were stained using DAPI (blue). Statistical evaluation of proximity ligation assays indicating increased interactions of INO80-V5 and YY1 (E, n = 3/3 animals, 3 independent wells per animal, two-tailed students t-test, *p<0.05) and of INO80-V5 to WDR5 (H, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, **p<0.01). No increase in proximity was detected between Ino80-V5 and Ruvbl2 (K, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, ns: not significant). Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays. No signals were detected for these controls. Data are mean ± S.E.M.


Assessment of mtDNA damage by quantitative PCR . HEK293 cells infected with control ( empty vector ) or shATG5 for 5 days . Cells were then incubated with DMSO or 4 mM 3 - NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h . Cells with or without washout were used for the extraction of total DNA . All DNA samples were used for amplification of 8 . 9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment . PCR products were quantitated by PicoGreen staining using Micro Plate Reader . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 . The data in ( A ) were further calculated for the frequency of mtDNA damage . The equation was seen in " Materials and Methods " . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a two - tailed student ' s t - test , N . S . , not significant , * * 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Assessment of mtDNA damage by quantitative PCR. HEK293 cells infected with control (empty vector) or shATG5 for 5 days. Cells were then incubated with DMSO or 4 mM 3-NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h. Cells with or without washout were used for the extraction of total DNA. All DNA samples were used for amplification of 8.9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment. PCR products were quantitated by PicoGreen staining using Micro Plate Reader. Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. The data in (A) were further calculated for the frequency of mtDNA damage. The equation was seen in "Materials and Methods". Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, **p < 0.01.


Representative images show 8 - oxo - dG staining . Control or shATG5 HeLa cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h , and then either fixed immediately or washed with fresh medium and incubated for another 1 h . Cells were immunostained with DAPI and anti - 8 - oxo - dG antibody and analyzed by confocal microscopy . Scale bar , 10 µm . Quantification of the relative 8 - oxo - dG fluorescence intensity in ( C ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by a two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 .

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

Representative images show 8-oxo-dG staining. Control or shATG5 HeLa cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and then either fixed immediately or washed with fresh medium and incubated for another 1 h. Cells were immunostained with DAPI and anti-8-oxo-dG antibody and analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in (C). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01.


Control or shATG5 HEK293 cells stably expressing mito - Keima . Control cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h , and shATG5 cells were treated with 200 µM H2O2 as a negative control . Cells were then imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Right panels show the pixel intensity of red ( mitochondria within lysosomes ) and green ( mitochondria in the cytoplasm ) from a line . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561 nm / 458 nm ) of the cells described in ( E ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by a two - way ANOVA , N . S . , not significant , * * p < 0 . 01 .

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

Control or shATG5 HEK293 cells stably expressing mito-Keima. Control cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and shATG5 cells were treated with 200 µM H2O2 as a negative control. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Right panels show the pixel intensity of red (mitochondria within lysosomes) and green (mitochondria in the cytoplasm) from a line. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (E). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-way ANOVA, N.S., not significant, **p < 0.01.


HeLa cells expressing GFP - LC3 were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h . Cells were stained with anti - Tom20 and anti - DNA antibodies , then analyzed by confocal microscopy . The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA / Tom20 . Scale bar , 10 µm . Quantification of the LC3 punta colocalizing or contacting with mtDNA / Tom20 in a cell . n = 30 cells from 3 coverslips , Data are presented as mean ± SD ( n = 30 ) , statistical significance was assessed by a one - way ANOVA with Tukey ' s multiple comparisons test , * * * p < 0 . 001 .

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

HeLa cells expressing GFP-LC3 were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were stained with anti-Tom20 and anti-DNA antibodies, then analyzed by confocal microscopy. The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA/Tom20. Scale bar, 10 µm. Quantification of the LC3 punta colocalizing or contacting with mtDNA/Tom20 in a cell. n = 30 cells from 3 coverslips, Data are presented as mean ± SD (n = 30), statistical significance was assessed by a one-way ANOVA with Tukey's multiple comparisons test, ***p < 0.001.


Screening of mitophagy regulators for removing damaged mtDNA . HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors . Five days later , cells were treated with 200 µM H2O2 for 2 h . Cells were then imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima . ShATG5 was used as a negative control . Right panels for each image show the FACS - based mito - Keima dot plots . The y - axis represents the fluorescence emission of mito - Keima at pH 4 . 0 ( lysosome ) , while the x - axis indicates mito - Keima at pH 7 . 0 ( mitochondria ) . The percentages of cells within the different regions are indicated . Scale bar , 10 µm .

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

Screening of mitophagy regulators for removing damaged mtDNA. HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors. Five days later, cells were treated with 200 µM H2O2 for 2 h. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima. ShATG5 was used as a negative control. Right panels for each image show the FACS-based mito-Keima dot plots. The y-axis represents the fluorescence emission of mito-Keima at pH 4.0 (lysosome), while the x-axis indicates mito-Keima at pH 7.0 (mitochondria). The percentages of cells within the different regions are indicated. Scale bar, 10 µm.


Control or ATAD3B KO HeLa cells stably expressing mito - Keima were treated with 4 mM 3 - NPA for 2 h and imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561 nm / 458 nm ) of the cells described in ( B ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

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

Control or ATAD3B KO HeLa cells stably expressing mito-Keima were treated with 4 mM 3-NPA for 2 h and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (B). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.


Control or KO ATAD3B HeLa cells were treated with 4 mM 3 - NPA for 2 h . Cells were washed with fresh medium and incubated for another 1 h . Cells were then fixed and immunostained with DAPI and anti - 8 - oxo - dG antibodies and were analyzed by confocal microscopy . Scale bar , 10 µm . Quantification of the relative 8 - oxo - dG fluorescence intensity in cells described in ( D ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

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

Control or KO ATAD3B HeLa cells were treated with 4 mM 3-NPA for 2 h. Cells were washed with fresh medium and incubated for another 1 h. Cells were then fixed and immunostained with DAPI and anti-8-oxo-dG antibodies and were analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in cells described in (D). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test , N.S., not significant, **p < 0.01.


PINK1 KO HeLa cells stably expressing mito - Keima were infected with control or shATAD3B . Five days later , cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h . Cells were then analyzed and imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561nm / 458nm ) of the cells described in ( A ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PINK1 KO HeLa cells stably expressing mito-Keima were infected with control or shATAD3B. Five days later, cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were then analyzed and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561nm/458nm) of the cells described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.


HeLa cells were treated with DMSO , 4 mM 3 - NPA , 8 mM 3 - NPA , or 10 µM CCCP for 2 h , and cell lysates were then analyzed by Western blotting using the indicated antibodies .

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

HeLa cells were treated with DMSO, 4 mM 3-NPA, 8 mM 3-NPA, or 10 µM CCCP for 2 h, and cell lysates were then analyzed by Western blotting using the indicated antibodies.


PINK1 KO HeLa cells stably expressing ATAD3A - Flag ( 3A - Flag ) or ATAD3B - Flag ( 3B - Flag ) were mixed with control PINK1 KO cells respectively , and incubated for 24 h . Cells were treated with 4 mM 3 - NPA ( D ) or 200 µM H2O2 ( F ) for 2 h , and then washed with fresh medium and incubated for another 1 h . Cells were fixed and immunostained with anti - 8 - oxo - dG and anti - Flag antibodies and analyzed by confocal microscopy . 8 - oxo - dG fluorescence intensity of cells treated with 3 - NPA ( E ) or H2O2 ( G ) was quantified by ImageJ software . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by individual two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 . Scale bar , 10 µm .

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

PINK1 KO HeLa cells stably expressing ATAD3A-Flag (3A-Flag) or ATAD3B-Flag (3B-Flag) were mixed with control PINK1 KO cells respectively, and incubated for 24 h. Cells were treated with 4 mM 3-NPA (D) or 200 µM H2O2 (F) for 2 h, and then washed with fresh medium and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and anti-Flag antibodies and analyzed by confocal microscopy. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (E) or H2O2 (G) was quantified by ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by individual two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. Scale bar, 10 µm.


Control ( GFP + ) and shATAD3B ( GFP - ) PINK1 KO HeLa cells were mixed and incubated for 24 h . Cells were then treated with 4 mM 3 - NPA ( H ) or 200 µM H2O2 ( J ) for 2 h , and washed with fresh medium , and incubated for another 1 h . Cells were fixed and immunostained with anti - 8 - oxo - dG and DAPI , then analyzed by confocal microscopy . Control or shATAD3B cells are circled by white dashed lines . 8 - oxo - dG fluorescence intensity of cells treated with 3 - NPA ( I ) or H2O2 ( K ) was quantified using ImageJ software . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , * * p < 0 . 01 . Scale bar , 10 µm .

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

Control (GFP+) and shATAD3B (GFP-) PINK1 KO HeLa cells were mixed and incubated for 24 h. Cells were then treated with 4 mM 3-NPA (H) or 200 µM H2O2 (J) for 2 h, and washed with fresh medium, and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and DAPI, then analyzed by confocal microscopy. Control or shATAD3B cells are circled by white dashed lines. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (I) or H2O2 (K) was quantified using ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, **p < 0.01. Scale bar, 10 µm.


293T cells were treated with H2O2 for 2 h , and cell lysates were then immunoprecipitated with Dynabeads Protein G pre - coupled with rabbit IgG ( control ) or anti - ATAD3 antibody , followed by Western blotting with anti - LC3B or anti - ATAD3 antibodies .

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

293T cells were treated with H2O2 for 2 h, and cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with rabbit IgG (control) or anti-ATAD3 antibody, followed by Western blotting with anti-LC3B or anti-ATAD3 antibodies.


ATAD3 DKO HeLa cells expressing GFP - LC3B were transiently transfected with control , ATAD3A - Flag or ATAD3B - Flag . 48 h after transfection , cell lysates were immunoprecipitated ( IP ) with anti - Flag M2 affinity gel , and analyzed by Western blotting using anti - Flag or anti - GFP antibodies .

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

ATAD3 DKO HeLa cells expressing GFP-LC3B were transiently transfected with control, ATAD3A-Flag or ATAD3B-Flag. 48 h after transfection, cell lysates were immunoprecipitated (IP) with anti-Flag M2 affinity gel, and analyzed by Western blotting using anti-Flag or anti-GFP antibodies.


HeLa cells were treated with 200 µM H2O2 for 2 h , and cell lysates were incubated with the indicated GST - ATAD3A or GST - ATAD3B protein fragments ( expressed in E . coli ) coupled to glutathione agarose beads ( Pierce ) for GST pull - down assay . Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B .

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

HeLa cells were treated with 200 µM H2O2 for 2 h, and cell lysates were incubated with the indicated GST-ATAD3A or GST-ATAD3B protein fragments (expressed in E.coli) coupled to glutathione agarose beads (Pierce) for GST pull-down assay. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.


The indicated GST - ATAD3A or GST - ATAD3B protein fragments and His - LC3B were expressed in E . coli and purified . GST pull - down assay was performed using glutathione agarose beads . Eluted protein samples were analyzed by Western blotting using anti - GST or anti - His antibodies .

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

The indicated GST-ATAD3A or GST-ATAD3B protein fragments and His-LC3B were expressed in E.coli and purified. GST pull-down assay was performed using glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using anti-GST or anti-His antibodies.


The indicated GST - ATAD3B ( WT ) or GST - ATAD3B ( LIR mutations ) and His - LC3 were expressed in E . coli and purified . GST pull - down assay was performed using the glutathione agarose beads . Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B .

The indicated GST-ATAD3B(WT) or GST-ATAD3B (LIR mutations) and His-LC3 were expressed in E.coli and purified. GST pull-down assay was performed using the glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.


HeLa expressing GFP - LC3 monoclonal cell line was infected with lentivirus particles containing control or shATAD3B . Five days later , cells were treated with DMSO , or 200 µM H2O2 for 2 h . Cells were fixed and immunostained with anti - Tom20 and imaged by confocal microscopy . The white arrows indicate the LC3 puncta colocalizing or contacting with Tom20 ( mitochondria ) . Scale bar , 10 µm .

O O O O O O O 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

HeLa expressing GFP-LC3 monoclonal cell line was infected with lentivirus particles containing control or shATAD3B. Five days later, cells were treated with DMSO, or 200 µM H2O2 for 2 h. Cells were fixed and immunostained with anti-Tom20 and imaged by confocal microscopy. The white arrows indicate the LC3 puncta colocalizing or contacting with Tom20 (mitochondria). Scale bar, 10 µm.