( C ) Active BAX is present in a subset of LC3 ‐ positive . Sections from P7 P14 MCL ‐ 1Δ / Δ brains were stained for LC3 ( Green ) active BAX ( 6A7 ; Red ) . Confocal were taken using × 63 objective . Arrows point to BAX ‐ positive , LC3 ‐ negative . Representative are shown . Scale bars = 50 μm .

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(C) Active BAX is present in a subset of LC3‐positive cells. Sections from P7 and P14 MCL‐1Δ/Δ brains were stained for LC3 (Green) and active BAX (6A7; Red). Confocal images were taken using × 63 objective. Arrows point to BAX‐positive, LC3‐negative cells. Representative images are shown. Scale bars=50 μm.


cortical neurons of indicated genotypes were infected with a lentivirus expressing Cre at time of plating analysed for presence of caspase ‐ fragment of caspase ‐ 3 substrate CAS5 days later . Alternatively ( E ) , neurons were analysed for cytochrome c release by immunofluorescence . Data are expressed as of positive for at least three per genotype ± s . d .

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Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate CAS5 days later. Alternatively (E), the neurons were analysed for cytochrome c release by immunofluorescence. Data are expressed as percent of positive cells for at least three embryos per genotype±s.d.


( A ) Brains from E12 . 5 Foxg1 Cre MCL ‐ 1 were analysed by western blot for presence of caspase ‐ fragment of caspase ‐ 3 substrate p130CAS autophagic markers LC3 p62 .

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(A) Brains from E12.5 Foxg1 Cre MCL‐1 animals were analysed by western blot for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate p130CAS and the autophagic markers LC3 and p62.


( B ) Autophagy in Foxg1 Cre MCL ‐ 1Δ / Δ mice . Brain sections from E15 . 5 with indicated genotypes were stained for LC3 ( green ) . Confocal were taken using × 20 ( bottom panel ) or × 63 ( top middle panels ) objectives . Scale bars = 50 μm .

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(B) Autophagy in Foxg1 Cre MCL‐1Δ/Δ mice. Brain sections from E15.5 animals with the indicated genotypes were stained for LC3 (green). Confocal images were taken using × 20 (bottom panel) or × 63 (top and middle panels) objectives. Scale bars=50 μm.


( C ) Colocalization of LC3 vesicles with lysosomal marker LAMP1 was analysed by immunohistochemistry in E15 . 5 wild ‐ type . Data are expressed as of LC3 vesicles that are LAMP1 ‐ positive ± s . d . About 100 LC3 vesicles were counted per section , with four sections per . VZ , ventricular zone .

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(C) Colocalization of LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunohistochemistry in E15.5 wild‐type embryos. Data are expressed as the percentage of LC3 vesicles that are LAMP1‐positive±s.d. About 100 LC3 vesicles were counted per section, with four sections per embryo. VZ, ventricular zone.


( D ) cortical neurons neuronal progenitor were analysed for presence of indicated apoptosis ( BIM , Puma ) autophagy ( ATG7 , ATG5 ‐ ATG12 ) related as well as Tuj ‐ 1 ( neuronal marker ) , Nestin ( neural progenitor marker ) HSC70 ( loading control ) .

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(D) Primary cortical neurons and neuronal progenitor cells were analysed for the presence of the indicated apoptosis (BIM, Puma) and autophagy (ATG7, ATG5‐ATG12) related proteins as well as Tuj‐1 (neuronal marker), Nestin (neural progenitor marker) and HSC70 (loading control).


( E ) neurospheres were treated for 8 h with EBSS analysed by western blot for presence of MCL ‐ 1 actin as a loading control .

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(E) Primary neurospheres were treated for 8 h with EBSS and analysed by western blot for the presence of MCL‐1 and actin as a loading control.


( F ) neurons neurospheres were treated as in E analysed by western blot for p130CAS , LC3 actin as a loading control .

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(F) Primary neurons and neurospheres were treated as in E and analysed by western blot for p130CAS, LC3 and actin as a loading control.


Alternatively , they were analysed by immunofluorescence for c release ( G ) in presence of 50 μM zVAD ‐ FMK to prevent apoptotic from detaching from coverslip . Data are expressed as average of 3 - 7 ± s . d . * P0 . 001 .

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Alternatively, they were analysed by immunofluorescence for c release (G) in the presence of 50 μM zVAD‐FMK to prevent apoptotic cells from detaching from the coverslip. Data are expressed as the average of 3-7 embryos±s.d. *P0.001.


( A ) Brain of P14 MCL ‐ 1Δ / Δ Beclin ‐ 1 + / − mice . Brain sections were stained with cresyl violet imaged using a × 1 objective .

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(A) Brain morphology of P14 MCL‐1Δ/Δ Beclin‐1+/− mice. Brain sections were stained with cresyl violet and imaged using a × 1 objective.


( B - D ) Active BAX ( 6A7 ; B ) active caspase ‐ 3 ( C ) were quantified by cortex of 14 ‐ day ‐ old MCL ‐ 1Δ / Δ MCL ‐ 1Δ / Δ Beclin - 1 + / − mice . number of BAX ‐ positive LC3 ‐ negative was also quantified . ( D ) Representative from at least three sections from each were counted . total number of was determined by counting Hoechst ‐ positive nuclei . Data are expressed as of positive for least three per genotype ± s . d . * P0 . 05 .

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(B-D) Active BAX (6A7; B) and active caspase‐3 (C) were quantified by staining the cortex of 14‐day‐old MCL‐1Δ/Δ and MCL‐1Δ/Δ Beclin-1+/− mice. The number of BAX‐positive LC3‐negative cells was also quantified. (D)Representative images from at least three sections from each animal were counted. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of positive cells for least three animals per genotype±s.d. *P0.05.


( E ) Survival data from MCL ‐ 1Δ / Δ ( n = 14 ) their MCL ‐ 1Δ / Δ Beclin ‐ 1 + / − ( n = 13 ) .

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(E) Survival data from MCL‐1Δ/Δ (n=14) and their MCL‐1Δ/Δ Beclin‐1+/− littermates (n=13).


Confocal demonstrate overlap of GFP tagged mtON with MitoTrackerTM Red CMXRos stained C . elegans hypodermal mitochondria . Scale bar 10 µm .

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Confocal images demonstrate overlap of GFP tagged mtON with MitoTrackerTM Red CMXRos stained C. elegans hypodermal mitochondria. Scale bar 10 µm.


Immunoblot comparing supernatant mitochondria - enriched pellet of isolation fractions . GFP - tagged mtON migrates at predicted molecular of 82 kDa accounting for mitochondria - target , proton pump , GFP . mtON is observed only in mitochondrial fraction compared to marker HSP60 ( mitochondria ) actin ( cytosol ) . blots are from same lanes on one .

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Immunoblot comparing the cytosolic supernatant and the mitochondria-enriched pellet of isolation fractions. GFP-tagged mtON migrates at the predicted molecular weight of 82 kDa accounting for the mitochondria-target sequence, the proton pump, and GFP. mtON is observed only in the mitochondrial fraction compared to marker proteins HSP60 (mitochondria) and actin (cytosol). All blots are from the same lanes on one membrane.


Representative TMRE traces in arbitrary units ( a . u . ) before after Δψm dissipation with FCCP . Dashed lines indicate where FCCP was added . + / - ATR traces were performed in absence of succinate . mtON activation was continuous throughout traces . green trace is from mitochondria with ATR , gray traces are without . of change in ( ΔF ) normalized to maximum change in given by succinate respiration ( ΔFmax ) for each mitochondrial preparation . Data are from maximum dose in Fig . EV 3A . Two - way ANOVA with Sidak ' s for multiple comparisons , * p = 0 . 0469 , + ATR succinate vs . + ATR + p = 0 . 9978 , n = 6 mitochondrial isolations . Data show mean ± .

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Representative TMRE fluorescence traces in arbitrary units (a.u.) before and after Δψm dissipation with FCCP. Dashed lines indicate where FCCP was added. +/- ATR traces were performed in the absence of succinate. mtON activation was continuous throughout the traces. Light green trace is from mitochondria with ATR, gray traces are without. Quantification of change in fluorescence (ΔF) normalized to the maximum change in fluorescence given by succinate respiration (ΔFmax) for each mitochondrial preparation. Data are from the maximum light dose in Fig. EV 3A. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0469, +ATR succinate vs. +ATR +light p = 0.9978, n = 6 mitochondrial isolations. Data show mean ± SEM.


Representative BCECF - AM 490 / 440 nm trace . of 545 nm at either 440 or 490 nm excitation . Dashed lines are where or FCCP treatment occurred . green trace is from mitochondria with ATR , gray traces are without . of change in BCECF - AM normalized to maximum change given by succinate matrix alkalization . Two - way ANOVA with Sidak ' s for multiple comparisons , - ATR succinate vs . - ATR , + * p = 0 . 0212 , + ATR succinate vs . + ATR p = 0 . 999 , + ATR succinate vs . + ATR , - p = 0 . 0237 , + ATR , + vs , + ATR , - p = 0 . 0474 , n = 3 mitochondrial isolations . Some points overlap . Data show mean ± .

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Representative BCECF-AM 490/440 nm ratio trace. Ratio of 545 nm fluorescence intensity at either 440 or 490 nm excitation. Dashed lines are where light or FCCP treatment occurred. Light green trace is from mitochondria with ATR, gray traces are without. Quantification of change in BCECF-AM fluorescence ratio normalized to maximum change given by succinate matrix alkalization. Two-way ANOVA with Sidak's test for multiple comparisons, -ATR succinate vs. -ATR, +light *p = 0.0212, +ATR succinate vs. +ATR light p = 0.999, +ATR succinate vs. +ATR,-light p = 0.0237, +ATR, +light vs, +ATR, -light p = 0.0474, n = 3 mitochondrial isolations. Some individual points overlap. Data show mean ± SEM.


ATP normalized to total ATP synthesis given by succinate respiration . Data from Fig . EV 3B . Succinate data shown for comparison after normalization . Two - way ANOVA with Sidak ' s for multiple comparisons , * p = 0 . 0011 , + ATR succinate vs + ATR p = 0 . 5680 , n = 3 , 7 , 3 , 7 , biological replicates for each bar from left to right . Some points overlap . Data show mean + .

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ATP levels normalized to total ATP synthesis given by succinate respiration. Data from Fig. EV 3B. Succinate data shown for comparison after normalization. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0011, +ATR succinate vs +ATR light p = 0.5680, n = 3,7,3,7, biological replicates for each bar from left to right. Some individual points overlap. Data show mean + SEM.


O2 required to consume 50 nmoles of ADP after mtON activation . Data are maximum illumination from Fig . EV 4D using time - matched conditions , One - way ANOVA * p = 0 . 013 ( - ATR , - vs . + ATR , + p = 0 . 010 . - ATR , + vs . + ATR , + p = 0 . 013 . ) - ATR , - n = 5 , n = 6 mitochondrial preparations . Data show mean + .

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O2 required to consume 50 nmoles of ADP after mtON activation. Data are the maximum illumination from Fig. EV 4D using time-matched dark conditions, One-way ANOVA *p = 0.013 (-ATR, -light vs. +ATR, +light p = 0.010. -ATR, +light vs. +ATR, +light p = 0.013.) -ATR, -light n = 5, rest n = 6 mitochondrial preparations. Data show mean + SEM.


Day 1 adult expressing mtON were exposed to 50 µM rotenone ( ETC I inhibitor ) for 5 hours survival was scored . Illumination was continuous throughout toxin exposure ( see methods ) . ATR alone was protective ( - ATR , - vs . + ATR , - p = 0 . 016 . ) effect of mtON activation was greater than ATR alone effect , one - way ANOVA with Tukey ' s post hoc * p = 0 . 01 . ( - ATR , - vs + ATR , + p = 0 . 0002 . - ATR , + vs . + ATR , + p = 0 . 0009 . n = 3 plates each condition with at least 15 per plate ) . Data show mean ± . mtON - expressing were exposed to 50 µM antimycin A ( III inhibitor ) survival was scored 18 hours later . One - way ANOVA with Tukey ' s post hoc * p = 0 . 02 ( - ATR , - vs + ATR , + p = 0 . 03 , - ATR , + vs . + ATR , + p = 0 . 01 . n = 5 plates each condition with at least 15 per plate ) . Data show mean ± . mtON - expressing were exposed to 0 . 25 M azide ( IV inhibitor ) for 1 hour scored for survival 1 hour after recovering , one - way ANOVA with Tukey ' s post hoc * p = 0 . 0002 . ( - ATR , - vs + ATR , + p < 0 . 0001 , - ATR , + vs . + ATR , + p < 0 . 0001 . n = 3 plates each condition with at least 15 per plate ) . Data show mean ± . mtON - expressing were exposed to 31 µM oligomycin A ( ATP synthase inhibitor ) survival was scored 18 hours later . No significant differences were found by one - way ANOVA ( n = 3 plates each condition with at least 15 per plate ) . Data show mean ± .

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Day 1 adult animals expressing mtON were exposed to 50 µM rotenone (ETC complex I inhibitor) for 5 hours and survival was scored. Illumination was continuous throughout toxin exposure (see methods). ATR alone was protective (-ATR, -light vs. +ATR, -light p = 0.016.) The effect of mtON activation was greater than the ATR alone effect, one-way ANOVA with Tukey's post hoc test *p = 0.01. (-ATR, -light vs +ATR, +light p = 0.0002. -ATR, +light vs. +ATR, +light p = 0.0009. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 50 µM antimycin A (complex III inhibitor) and survival was scored 18 hours later. One-way ANOVA with Tukey's post hoc test *p = 0.02 (-ATR, -light vs +ATR, +light p = 0.03, -ATR, +light vs. +ATR, +light p = 0.01. n = 5 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 0.25 M azide (complex IV inhibitor) for 1 hour and scored for survival 1 hour after recovering, one-way ANOVA with Tukey's post hoc test *p = 0.0002. (-ATR, -light vs +ATR, +light p < 0.0001, -ATR, +light vs. +ATR, +light p <0.0001. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 31 µM oligomycin A (ATP synthase inhibitor) and survival was scored 18 hours later. No significant differences were found by one-way ANOVA (n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM.


mtON - expressing in a I mutant background ( - 1 ) were scored for locomotion speed in presence of food by counting bends per minute . mtON activation rescued decreased locomotion of - 1 mutant background , one - way ANOVA with Tukey ' s post hoc , - ATR , + vs . + ATR , + * p = 0 . 0145 , + ATR , + vs . wildtype ( WT ) * p = 0 . 0008 ( - ATR , - vs + ATR , + p = 0 . 0272 , + ATR , - vs . + ATR , + p = 0 . 0332 , conditions vs WT , p < 0 . 001 , n = 30 , 30 , 30 , 41 , 41 each bar from left to right ) . Data show mean ± standard deviation .

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mtON-expressing animals in a complex I mutant background (gas-1) were scored for locomotion speed in the presence of food by counting body bends per minute. mtON activation rescued the decreased locomotion of the gas-1 mutant background, one-way ANOVA with Tukey's post hoc test, -ATR, +light vs. +ATR, +light *p = 0.0145, +ATR, +light vs. wildtype (WT) *p = 0.0008 (-ATR, -light vs +ATR, +light p = 0.0272, +ATR, -light vs. +ATR, +light p = 0.0332, all conditions vs WT, p < 0.001, n = 30, 30,30,41,41 animals each bar from left to right). Data show mean ± standard deviation.


Top : Immunoblot assessing effect of mtON activation on AMPK phosphorylation status . Top bands ( ~ 62 kDa ) are phosphorylated AMPK signal , bottom bands ( ~ 43 kDa ) are actin signal . Image is from same cut to separately probe for phosphorylated AMPK ( pAMPK ) actin . Bottom : Densitometry showing decreased pAMPK to actin , as there is no known antibody directed against total AMPK in C . elegans . Phosphorylation increases in absence of food , but low phosphorylation is preserved when mtON is activated , two - way ANOVA with Sidak ' s for multiple comparisons , * p = 0 . 0203 , n = 3 , 3 , 3 , 4 , 4 biological replicates each bar from left to right . Data show mean + standard deviation .

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Top: Immunoblot assessing the effect of mtON activation on AMPK phosphorylation status. Top bands (~62 kDa) are phosphorylated AMPK signal, and bottom bands (~43 kDa) are actin signal. Image is from the same membrane cut to separately probe for phosphorylated AMPK (pAMPK) and actin. Bottom: Densitometry analysis showing decreased pAMPK to actin ratio, as there is no known antibody directed against total AMPK in C. elegans. Phosphorylation increases in the absence of food, but low phosphorylation is preserved when mtON is activated, two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0203, n = 3,3,3,4,4 biological replicates each bar from left to right. Data show mean + standard deviation.


Locomotion was assessed by counting bends per minute . Wild type were compared to aak - 2 ( ok524 ) mutant . 2 - sample , 2 - unpaired t - * p < 0 . 0001 , wild type n = 35 , aak - 2 n = 39 across at least 3 days . Data show mean ± standard deviation .

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Locomotion was assessed by counting body bends per minute. Wild type animals were compared to aak-2(ok524) mutant animals. 2-sample, 2-tailed unpaired t-test *p < 0.0001, wild type n = 35, aak-2 n = 39 animals across at least 3 days. Data show mean ± standard deviation.


Locomotion in response to mtON activation . Illumination was continuous throughout bends measurement ( see methods ) . For AAK - 2 activation , were exposed to 1 mM AICAR for 4 hours before bends measurement . One - way ANOVA with Tukey ' s post hoc , - ATR , + vs + ATR , + * p < 0 . 0001 , + ATR , + vs . AICAR * p < 0 . 0001 , + ATR , + vs AICAR + * p < 0 . 0001 . n = 36 , 39 , 37 , 46 , 36 , 36 each bar from left to right . Data show mean ± standard deviation .

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Locomotion in response to mtON activation. Illumination was continuous throughout body bends measurement (see methods). For AAK-2 activation, animals were exposed to 1 mM AICAR for 4 hours before body bends measurement. One-way ANOVA with Tukey's post hoc test, -ATR, +light vs +ATR, +light *p < 0.0001, +ATR, +light vs. AICAR *p < 0.0001, +ATR,+light vs AICAR +light *p < 0.0001. n = 36, 39, 37, 46, 36, 36 animals each bar from left to right. Data show mean ± standard deviation.


Survival after HR in day 1 adult is shown with hypoxic preconditioning ( PC , represented by diagonal stripes ) 2 - sample 2 - unpaired t - , ctrl vs . PC * p = 0 . 0035 , n = 4 , each averaged from 3 technical replicates with at least 15 per replicate . Data show mean ± .

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Survival after HR in day 1 adult animals is shown with hypoxic preconditioning (PC, represented by diagonal stripes) 2-sample 2-tailed unpaired t-test, ctrl vs. PC *p = 0.0035, n = 4, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Protection ( % ) is survival minus survival of control condition . PC data calculated from panel B . FCCP final concentrations 0 . 001 , 0 . 01 , 0 . 1 , 1 nM . One - way ANOVA with Tukey ' s post hoc , PC vs . 0 . 001 nM FCCP , * p = 0 . 0012 , 0 . 001 nM FCCP vs 0 . 01 FCCP , * p = 0 . 0142 , n = 4 , 3 , 3 , 3 , 3 independent experiments each bar from left to right , each averaged from 3 technical replicates with at least 15 per replicate . Data show mean ± .

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Protection (%) is percent survival minus percent survival of control condition. PC data calculated from panel B. FCCP final concentrations 0.001, 0.01, 0.1, 1 nM. One-way ANOVA with Tukey's post hoc test, PC vs. 0.001 nM FCCP, *p = 0.0012, 0.001 nM FCCP vs 0.01 FCCP, *p = 0.0142, n = 4,3,3,3,3 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Illumination was continuous throughout PC alone ; control illumination was for same duration under normoxic conditions ( see methods & panel A ) . Two - way ANOVA comparing + ATR vs - ATR in each group . * p = 0 . 016 , n = 11 , 11 , 4 , 4 , 6 , 6 independent experiments each bar from left to right , each averaged from 3 technical replicates with at least 15 per replicate . Data show mean ± .

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Illumination was continuous throughout PC alone; control illumination was for the same duration under normoxic conditions (see methods & panel A). Two-way ANOVA comparing +ATR vs -ATR in each group. *p = 0.016, n = 11,11,4,4,6,6 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


during hypoxia . 2 sample 2 - unpaired t - , * p = 0 . 0276 , n = 7 independent experiments , each averaged from 3 technical replicates with at least 15 per replicate . Data show mean ± .

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light during hypoxia. 2 sample 2-tailed unpaired t-test, *p = 0.0276, n = 7 independent experiments, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


A , Rb1 ( RB ) was expressed in uterine luminal epithelium stroma during early pregnancy in mice . Scale bar = 100μm ; arrowhead , . B , Phosphorylated RB ( pRB ) , inactive form of RB , was expressed in luminal epithelium on days 2 3 of pregnancy . Scale bar = 100μm .

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A, Rb1 protein (RB) was expressed in uterine luminal epithelium and stroma during early pregnancy in mice. Scale bar = 100μm; arrowhead, embryo. B, Phosphorylated RB (pRB) protein, the inactive form of RB, was expressed in the luminal epithelium on days 2 and 3 of pregnancy. Scale bar = 100μm.


C , RB was efficiently deleted in uterus of mice with uterine deletion of Rb1 ( Rb1d / d mice ) on day 4 of pregnancy . Scale bar = 100μm . D , RB expression was normal in ovary of Rb1d / d mice on day 4 of pregnancy . Scale bar = 100μm .

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C, RB was efficiently deleted in the uterus of mice with uterine deletion of Rb1 (Rb1d/d mice) on day 4 of pregnancy. Scale bar = 100μm. D, RB expression was normal in the ovary of Rb1d/d mice on day 4 of pregnancy. Scale bar = 100μm.


E , number of new - born pups was decreased in Rb1d / d dams ( n = 37 different dams ) compared to their littermate controls ( Rb1f / f mice , n = 20 different dams ) ( mean ± , Student ' s t ) .

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E, The number of new-born pups was decreased in Rb1d/d dams (n=37 different dams) compared to their littermate controls (Rb1f/f mice, n=20 different dams) (mean ± SEM, Student's t test).


A - C , Ovulation , fertilization development of preimplantation were normal in Rb1d / d mice ( A & C , mean ± , Student ' s t ; n = 5 mice for each group ; B , Fisher ' s exact ; n = 5 mice for each group ) .

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A-C, Ovulation, fertilization and development of preimplantation embryos were normal in Rb1d/d mice (A&C, mean ± SEM, Student's t test; n=5 mice for each group; B, Fisher's exact test; n=5 mice for each group).


Number of Ki67 - positive on day 4 in uterine epithelium of Rb1d / d mice was higher than that of Rb1f / f mice ( mean ± , Student ' s t ; n = 5 mice for each group ) . Scale bar , 100μm ; le , luminal epithelium ; s , stroma .

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Number of Ki67-positive cells on day 4 in uterine epithelium of Rb1d/d mice was higher than that of Rb1f/f mice (mean ± SEM, Student's t test; n=5 mice for each group). Scale bar, 100μm; le, luminal epithelium; s, stroma.


F - G , attachment occurred normally in Rb1d / d mice at 0900h on day 5 ( mean ± , Student ' s t ; n = 5 mice for each group ) . Arrow , implantation site ; scale bar = 1 cm .

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F-G, Embryo attachment occurred normally in Rb1d/d mice at 0900h on day 5 (mean ± SEM, Student's t test; n=5 mice for each group). Arrow, implantation site; scale bar = 1 cm.


H - I , Rb1d / d mice showed higher rate of resorption on day 8 ( * P < 0 . 05 ; Fisher ' s exact ; n = 45 implantation sites for Rb1f / f mice n = 46 implantation sites for Rb1d / d mice ) . Arrow , implantation site ; scale bar = 1 cm .

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H-I, Rb1d/d mice showed higher rate of embryo resorption on day 8 (*P<0.05; Fisher's exact test; n=45 implantation sites for Rb1f/f mice and n=46 implantation sites for Rb1d/d mice). Arrow, implantation site; scale bar = 1 cm.


J , H & E showed resorption in Rb1d / d mice on day 8 of pregnancy . Scale bar = 200 μm ; arrowhead , ; arrow , broken with blood infiltration .

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J, H&E staining showed embryo resorption in Rb1d/d mice on day 8 of pregnancy. Scale bar = 200 μm; arrowhead, embryo; arrow, broken embryo with blood cell infiltration.


K , of implantation site was comparable between Rb1f / f Rb1d / d mice on day 6 , but reduced on day 8 in Rb1d / d mice compared to Rb1f / f mice ( mean ± , Student ' s t ; n = 6 mice for each group ) .

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K, Weight of implantation site was comparable between Rb1f/f and Rb1d/d mice on day 6, but reduced on day 8 in Rb1d/d mice compared to Rb1f/f mice (mean ± SEM, Student's t test; n=6 mice for each group).


L - M , Expression of Cox2 Bmp2 , markers of decidualization , was comparable in between Rb1d / d Rb1f / f mice at 0900h on day 6 in implantation sites ( mean ± , Student ' s t ; n = 4 mice for each group ) .

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L-M, Expression of Cox2 and Bmp2, markers of decidualization, was comparable in between Rb1d/d and Rb1f/f mice at 0900h on day 6 in implantation sites (mean ± SEM, Student's t test; n=4 mice for each group).


A , Immunostaining of cytokeratin 8 ( CK8 ) , a marker of epithelial lineage , showed that epithelial alignment is collapsed trophoblast invades stroma at implantation sites in Rb1f / f mice , while epithelial alignment is persistent trophoblast invasion into stroma is compromised in Rb1d / d mice . Scale bar = 200μm ; arrowhead , an .

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A, Immunostaining of cytokeratin 8 (CK8), a marker of epithelial cell lineage, showed that epithelial alignment is collapsed and the trophoblast invades the stroma at implantation sites in Rb1f/f mice, while epithelial alignment is persistent and trophoblast invasion into the stroma is compromised in Rb1d/d mice. Scale bar = 200μm; arrowhead, an embryo.


B , Transmission electron microscopic analyses ( ) showed that uterine epithelium surrounding disappears trophoblast contacts directly with stroma at 2000h on day 5 in Rb1f / f mice , while uterine epithelium is persistent does not attach to stroma in Rb1d / d mice . Scale bar = 10µm ; red dotted , stroma ; green dotted , luminal epithelium ; blue dotted , trophoblast .

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B, Transmission electron microscopic analyses (TEM) showed that the uterine epithelium surrounding the embryo disappears and the trophoblast contacts directly with the stroma at 2000h on day 5 in Rb1f/f mice, while the uterine epithelium is persistent and the embryo does not attach to the stroma in Rb1d/d mice. Scale bar = 10µm; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


C , showed that fragmented luminal epithelial with lipid droplets are engulfed by trophoblast in control mice ( Rb1f / f mice ) . Scale bar = 2µm . Arrowhead , fragments engulfed by trophoblast ; yellow dotted circle , lipid droplets in luminal epithelium ; red dotted , stroma ; green dotted , luminal epithelium ; blue dotted , trophoblast .

C, TEM showed that fragmented luminal epithelial cells with cytoplasmic lipid droplets are engulfed by the trophoblast in the control mice (Rb1f/f mice). Scale bar = 2µm. Arrowhead, cytoplasmic fragments engulfed by trophoblast; yellow dotted line circle, cytoplasmic lipid droplets in the luminal epithelium; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


D , showed that microvilli , markers of uterine receptivity , were observed at surface of intact luminal epithelium on day 4 of pregnancy epithelial with lipid droplets were observed at 2000h on day 5 of pregnancy in control mice ( Rb1f / f mice ) . Scale bar = 2µm ; red dotted , stroma ; green dotted , luminal epithelium ; blue dotted , trophoblast .

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D, TEM showed that the microvilli, markers of uterine receptivity, were observed at the surface of intact luminal epithelium on day 4 of pregnancy and epithelial cells with cytoplasmic lipid droplets were observed at 2000h on day 5 of pregnancy in the control mice (Rb1f/f mice). Scale bar = 2µm; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


E , Immunofluorescence of MFG - E8 , a marker of programmed , showed that MFG - E8 is not expressed at implantation sites of Rb1f / f mice at 0900h on day 5 Rb1d / d mice at 0900h 2000h on day 5 , but is expressed at implantation sites of Rb1f / f mice at 2000h on day 5 . Scale bar = 100μm ; blue signal , nuclei stained by DAPI ; yellow signal , MFG - E8 ; arrowhead , .

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E, Immunofluorescence of MFG-E8, a marker of programmed cell death, showed that MFG-E8 is not expressed at the implantation sites of Rb1f/f mice at 0900h on day 5 and Rb1d/d mice at 0900h and 2000h on day 5, but is expressed at the implantation sites of Rb1f/f mice at 2000h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; arrowhead, embryo.


A , Serum estradiol - 17β ( E2 ) on days 4 6 of pregnancy were comparable between Rb1f / f Rb1d / d mice ( mean ± , Student ' s t ; n = 4 mice for each group ) .

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A, Serum estradiol-17β (E2) levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice (mean ± SEM, Student's t test; n=4 mice for each group).


B , Serum P4 on days 4 6 of pregnancy were comparable between Rb1f / f Rb1d / d mice . In Rb1d / d mice , pre - implantation P4 treatment on days 2 3 increased serum P4 on day 4 but did not affect those on day 6 ( mean ± , Student ' s t ; n = 4 mice for each group ) .

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B, Serum P4 levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice. In Rb1d/d mice, pre-implantation P4 treatment on days 2 and 3 increased serum P4 levels on day 4 but did not affect those on day 6 (mean ± SEM, Student's t test; n=4 mice for each group).


D , Persistent Ki67 expression in epithelium of Rb1d / d mice on day 4 was recovered by pre - implantation P4 treatment on days 2 3 . Scale bar = 100μm ; le , luminal epithelium ; s , stroma . E , of Ki67 - positive epithelial in Rb1d / d mice was completely suppressed by pre - implantation P4 supplementation on days 2 3 . same data of Rb1f / f Rb1d / d mice without P4 treatment in Fig 2D were used ( mean ± , Student ' s t ; n = 5 different mice for each group ) . Three different high - powered fields per mouse were analyzed each of Ki67 - posivie ratios was demonstrated .

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D, Persistent Ki67 expression in the epithelium of Rb1d/d mice on day 4 was recovered by pre-implantation P4 treatment on days 2 and 3. Scale bar = 100μm; le, luminal epithelium; s, stroma. E, Ratio of Ki67-positive epithelial cells in Rb1d/d mice was completely suppressed by pre-implantation P4 supplementation on days 2 and 3. The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2D were used (mean ± SEM, Student's t test; n=5 different mice for each group). Three different high-powered fields per mouse were analyzed and each of the Ki67-posivie ratios was demonstrated.


G , Resorption rate was reduced on day 8 in Groups 1 ( n = 41 different implantation sites ) 2 ( n = 30 different implantation sites ) , but not in Group 3 ( n = 40 different implantation sites ) . same data of Rb1f / f Rb1d / d mice without P4 treatment in Fig 2I were used . H & E was performed using sections that shows implantation sites , resorption rate was evaluated . * P < 0 . 05 vs Rb1f / f mice ; * * P < 0 . 05 vs Rb1d / d mice ; Fisher ' s exact .

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G, Resorption rate was reduced on day 8 in Groups 1 (n=41 different implantation sites) and 2 (n=30 different implantation sites), but not in Group 3 (n=40 different implantation sites). The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2I were used. H&E staining was performed using all sections that shows the implantation sites, and resorption rate was evaluated. *P<0.05 vs Rb1f/f mice; **P<0.05 vs Rb1d/d mice; Fisher's exact test.


H , Number of pups delivered by Rb1d / d dams was normalized by pre - implantation P4 treatment on days 2 3 ( Group 2 ) ( mean ± , Student ' s t ) . same data of Rb1f / f Rb1d / d dams without P4 treatment in Fig 1E were used . numbers of pups in 20 Rb1f / f 37 Rb1d / d dams without P4 treatment 7 Rb1d / d dams with P4 treatment were evaluated .

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H, Number of pups delivered by Rb1d/d dams was normalized by pre-implantation P4 treatment on days 2 and 3 (Group 2)(mean ± SEM, Student's t test). The same data of Rb1f/f and Rb1d/d dams without P4 treatment in Fig 1E were used. The numbers of pups in 20 Rb1f/f and 37 Rb1d/d dams without P4 treatment and 7 Rb1d/d dams with P4 treatment were evaluated.


A , pRIP3 , a central mediator of necroptosis , was not expressed at implantation sites of Rb1d / d mice but was expressed at those of Rb1f / f mice at 1500h on day 5 . Scale bar = 100μm ; blue signal , nuclei stained by DAPI ; purple signal , pRIP3 ; le , luminal epithelium ; s , stroma ; arrowhead , ; green dotted , luminal epithelium ; red dotted , stroma .

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A, pRIP3, a central mediator of necroptosis, was not expressed at the implantation sites of Rb1d/d mice but was expressed at those of Rb1f/f mice at 1500h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo; green dotted line, luminal epithelium; red dotted line, stroma.


B , TNFα stimulated expression of pMLKL , a critical mediator of necroptosis , in mouse uterine epithelial . Scale bar = 50μm ; blue signal , nuclei stained by DAPI ; red signal , pMLKL .

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B, TNFα stimulated the expression of pMLKL, a critical mediator of necroptosis, in the primary mouse uterine epithelial cells. Scale bar = 50μm; blue signal, nuclei stained by DAPI; red signal, pMLKL.


C , Uterine expression of Tnfα was elevated in control mice ( Rb1f / f mice ) on days 5 6 compared to on day 4 ( mean ± , Student ' s t ; n = 3 mice for each group ) . As for uterine samples on days 5 6 , those with implantation sites were examined .

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C, Uterine expression of Tnfα was elevated in the control mice (Rb1f/f mice) on days 5 and 6 compared to on day 4 (mean ± SEM, Student's t test; n=3 mice for each group). As for the uterine samples on days 5 and 6, those with implantation sites were examined.


D , implanting uterus produced TNFα at implantation site of both Rb1f / f Rb1d / d mice . Immunostaining of TNFα at implantation sites of Rb1f / f Rb1d / d mice were performed . Scale bar = 100μm ; arrowhead , .

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D, The implanting embryo and the uterus produced TNFα at the implantation site of both Rb1f/f and Rb1d/d mice. Immunostaining of TNFα at the implantation sites of Rb1f/f and Rb1d/d mice were performed. Scale bar = 100μm; arrowhead, embryo.


A - B , In vitro analyses using mouse uterine epithelial demonstrated that TNFα induces expression of annexin V , a marker of PS presentation at outer of Rb1f / f epithelial but does not in Rb1d / d ones , P4 restores TNFα - primed annexin V expression in Rb1d / d uterine epithelial ( mean ± , Student ' s t ) . Using three lines of mouse epithelial obtained from both Rb1f / fand Rb1d / d mice , in vivo experiments were performed three times . Each of annexin V - positive ratios was demonstrated . Scale bar = 100μm ; green signal , annexin V .

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A-B, In vitro analyses using primary mouse uterine epithelial cells demonstrated that TNFα induces the expression of annexin V, a marker of PS presentation at the outer membrane of Rb1f/f epithelial cells but does not in Rb1d/d ones, and P4 restores TNFα-primed annexin V expression in Rb1d/d uterine epithelial cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.


C - D , Annexin V using mouse uterine epithelial showed that TNFα administration increases expression of annexin V in growth - arrested mouse epithelial with thymidine treatment , but does not in control ( mean ± , Student ' s t ) . Using three lines of mouse epithelial obtained from both Rb1f / fand Rb1d / d mice , in vivo experiments were performed three times . Each of annexin V - positive ratios was demonstrated . Scale bar = 100μm ; green signal , annexin V .

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C-D, Annexin V assay using primary mouse uterine epithelial cells showed that TNFα administration increases the expression of annexin V in growth-arrested mouse epithelial cells with thymidine treatment, but does not in the control cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.


E , expression of TNF receptor type 2 ( TNFR2 ) was upregulated in growth - arrested epithelial with thymidine P4 supplementation ( mean ± , Student ' s t ) . Using three lines of mouse epithelial obtained from both Rb1f / fand Rb1d / d groups , qPCR was performed in duplicate .

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E, The expression of TNF receptor type 2 (TNFR2) was upregulated in growth-arrested epithelial cells with thymidine and P4 supplementation (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d groups, qPCR was performed in duplicate.


A , In Rb1d / d mice with pre - implantation P4 treatment , fragmented uterine epithelial with lipid droplets were engulfed by trophoblast . Scale bar = 2µm . Arrowhead , fragments engulfed by trophoblast ; dotted circle , lipid droplets in cytoplasm ; tr , trophoblast ; le , luminal epithelium ; s , stroma ; red dotted , stroma ; green dotted , luminal epithelium ; blue dotted , trophoblast .

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A, In Rb1d/d mice with pre-implantation P4 treatment, the fragmented uterine epithelial cells with the cytoplasmic lipid droplets were engulfed by the trophoblast cells. Scale bar = 2µm. Arrowhead, cytoplasmic fragments engulfed by trophoblast; dotted line circle, lipid droplets in the cytoplasm; tr, trophoblast; le, luminal epithelium; s, stroma; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


B , Pre - implantation P4 treatment rescued expression of MFG - E8 on day 5 of pregnancy in Rb1d / d uteri . Scale bar = 100μm . Blue signal , nuclei stained by DAPI ; yellow signal , MFG - E8 ; le , luminal epithelium ; s , stroma ; arrowhead , . C , Pre - implantation P4 treatment recovered expression of pRIP3 , a mediator of necroptosis , on day 5 of pregnancy in Rb1d / d uteri . Scale bar = 100μm ; blue signal , nuclei stained by DAPI ; purple signal , pRIP3 ; le , luminal epithelium ; s , stroma ; arrowhead , .

O O O O O O O O O O B-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 B-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

B, Pre-implantation P4 treatment rescued the expression of MFG-E8 on day 5 of pregnancy in Rb1d/d uteri. Scale bar = 100μm. Blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; le, luminal epithelium; s, stroma; arrowhead, embryo. C, Pre-implantation P4 treatment recovered the expression of pRIP3, a mediator of necroptosis, on day 5 of pregnancy in Rb1d/d uteri. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo.


A , RB was efficiently deleted in uterine epithelium of uterine epithelium - specific conditional knockout mice ( Rb1 eKO mice ) . Scale bar = 100μm . Rb1 eKO mice , Rb1loxP / loxPLtfCre / + mice ; Rb1 eControl mice ( littermate controls of Rb1 eKO mice ) , Rb1loxP / loxP mice ; le , luminal epithelium ; s , stroma .

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

A, RB was efficiently deleted in the uterine epithelium of uterine epithelium-specific conditional knockout mice (Rb1 eKO mice). Scale bar = 100μm. Rb1 eKO mice, Rb1loxP/loxPLtfCre/+ mice; Rb1 eControl mice (littermate controls of Rb1 eKO mice), Rb1loxP/loxP mice; le, luminal epithelium; s, stroma.


B , Number of new - born pups was comparable between Rb1 eKO eControl dams ( mean ± , Student ' s t ; n = 10 different dams for each group ) .

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B, Number of new-born pups was comparable between Rb1 eKO and eControl dams (mean ± SEM, Student's t test; n=10 different dams for each group).


C , expression patterns of Ki67 on day 4 in uterine epithelium were comparable between Rb1 eKO Rb1 eControl mice . Scale bar , 100μm ; le , luminal epithelium ; s , stroma .

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C, The expression patterns of Ki67 on day 4 in uterine epithelium were comparable between Rb1 eKO and Rb1 eControl mice. Scale bar, 100μm; le, luminal epithelium; s, stroma.


A , Representative pictures of Ki67 in human endometria obtained from women at time of implantation . Samples were divided into two groups according to outcome of clinical pregnancy following endometrial biopsy . Scale bar = 100μm ; le , luminal epithelium ; s , stroma . B , of Ki67 - positive in human uterine luminal epithelium during implantation period was lower in group with pregnancy than group without pregnancy ( mean ± , Student ' s t ; n = 10 different individuals for each group ) . Three different high - powered fields per sample were analyzed each of Ki67 - posivie ratios was demonstrated .

O 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 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 B-MEASURED_VAR O O O O O O

A, Representative pictures of Ki67 staining in the human endometria obtained from women at the time of embryo implantation. Samples were divided into two groups according to the outcome of clinical pregnancy following endometrial biopsy. Scale bar = 100μm; le, luminal epithelium; s, stroma. B, Ratio of Ki67-positive cells in the human uterine luminal epithelium during the implantation period was lower in the group with pregnancy than the group without pregnancy (mean ± SEM, Student's t test; n=10 different individuals for each group). Three different high-powered fields per sample were analyzed and each of the Ki67-posivie ratios was demonstrated.


( A ) Gross of control ( Ctrl ) , Sas - 4 − / − , Sas - 4 − / − 53bp1 − / − , Sas - 4 − / − Usp28 − / − at E9 . 5 . At least five were considered per genotype double mutant exhibited rescue criteria mentioned in text . Scale bar = 500 μm .

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

(A) Gross morphology of control (Ctrl), Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. At least five embryos were considered per genotype and all the double mutant embryos exhibited the rescue criteria mentioned in the text. Scale bar = 500 μm.


( B , C ) Immunostaining for p53 ( B ) - Caspase3 ( Cl - CASP3 , C ) on transverse sections of Ctrl , Sas - 4 − / − , Sas - 4 − / − 53bp1 − / − , Sas - 4 − / − Usp28 − / − at E9 . 5 . shown encompasses ventral neural tube surrounding mesenchyme . Asterisks in ( B ) denote non - specific of blood . Scale bar = 50 μm . ( D , E ) Quantifications of p53 ( D ) Cl - CASP3 ( E ) signals shown in ( B ) ( C ) , respectively . Three per genotype were used for quantifications . * * * p < 0 . 001 , * p < 0 . 05 ( two - student ' s T - , one - way ANOVA with Tukey ' s multiple comparisons gave similar results ) . Bars represent mean ± s . d . ( D ) Ctrl : 1 . 00 ± 0 . 06 ( n = 1770 ) ; Sas - 4 − / − : 6 . 84 ± 0 . 34 ( n = 275 ) , Sas - 4 − / − 53bp1 − / − : 1 . 85 ± 0 . 75 ( n = 690 ) , Sas - 4 − / − Usp28 − / − : 1 . 50 ± 0 . 48 ( n = 975 ) . ( E ) Ctrl : 1 . 00 ± 0 . 70 ( n = 1690 ) ; Sas - 4 − / − : 7 . 88 ± 2 . 62 ( n = 175 ) ; Sas - 4 − / − 53bp1 − / − : 1 . 43 ± 0 . 71 ( n = 1030 ) ; Sas - 4 − / − Usp28 − / − : 1 . 57 ± 0 . 44 ( n = 950 ) .

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

(B, C) Immunostaining for p53 (B) and Cleaved-Caspase3 (Cl-CASP3, C) on transverse sections of Ctrl, Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. The area shown encompasses the ventral neural tube and surrounding mesenchyme. Asterisks in (B) denote non-specific staining of blood cells. Scale bar = 50 μm. (D, E) Quantifications of the p53 (D) and Cl-CASP3 (E) fluorescence signals shown in (B) and (C), respectively. Three embryos per genotype were used for the quantifications. *** p < 0.001, *p<0.05 (two-tailed student's T-test, one-way ANOVA with Tukey's multiple comparisons tests gave similar results). Bars represent mean ± s.d. (D) Ctrl: 1.00 ± 0.06 (n=1770 cells); Sas-4−/−: 6.84 ± 0.34 (n=275), Sas-4−/− 53bp1−/−: 1.85 ± 0.75 (n=690), Sas-4−/− Usp28−/−: 1.50 ± 0.48 (n=975). (E) Ctrl: 1.00 ± 0.70 (n=1690); Sas-4−/−: 7.88 ± 2.62 (n=175); Sas-4−/− 53bp1−/−: 1.43 ± 0.71 (n=1030); Sas-4−/− Usp28−/−: 1.57 ± 0.44 (n=950).


( A , B ) Whole - mount immunostaining for p53 on Ctrl Sas - 4 − / − at E7 . 5 ( A ) E6 . 5 ( B ) . Representative sagittal planes are shown dotted lines demarcate epiblast . of p53 in epiblast is shown below , normalized to control in same batch at E7 . 5 ( 4 ) at E6 . 5 ( 9 ) . * * * p < 0 . 001 ( two - student ' s T - ) . Bars represent mean ± s . d . Scale bars = 100 μm . ( A ) Ctrl : 1 . 04 ± 0 . 00 ( n = 495 ) ; Sas - 4 - / - : 1 . 65 ± 0 . 19 ( n = 850 ) . ( B ) Ctrl : 1 . 00 ± 0 . 18 ( n = 1665 ) ; Sas - 4 - / - : 1 . 16 ± 0 . 44 ( n = 1330 )

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

(A, B) Whole-mount immunostaining and quantification for p53 on Ctrl and Sas-4−/− embryos at E7.5 (A) and E6.5 (B). Representative sagittal planes are shown and the dotted lines demarcate the epiblast. Quantification of nuclear p53 fluorescence intensity in the epiblast is shown below, normalized to control embryos in the same batch at E7.5 (4 embryos) and at E6.5 (9 embryos). *** p < 0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bars = 100 μm. (A) Ctrl: 1.04 ± 0.00 (n=495 cells); Sas-4-/-: 1.65 ± 0.19 (n=850). (B) Ctrl: 1.00 ± 0.18 (n=1665); Sas-4-/-: 1.16 ± 0.44 (n=1330)


( C , D ) Immunostaining for 53BP1 USP28 on sagittal sections of WT at E6 . 5 ( C ) or sagittal planes of whole - mount immunostaining at E5 . 5 ( D ) . of signals in epiblast ( Ep ) versus visceral endoderm ( VE ) are shown below . Four per genotype were used for quantifications . * * * * p < 0 . 0001 , * * * p < 0 . 001 ( two - student ' s T - ) . Bars represent mean ± s . d . Scale bar = 50 μm . ( C ) 53BP1 in Ep : 1 . 00 ± 0 . 10 ( n = 225 ) ; 53BP1 in VE : 0 . 37 ± 0 . 06 ( n = 105 ) ; USP28 in Ep : 1 . 00 ± 0 . 08 ( n = 235 ) ; USP28 in VE : 0 . 17 ± 0 . 05 ( n = 90 ) . ( D ) 53BP1 in Ep : 1 . 00 ± 0 . 24 ( n = 225 ) ; 53BP1 in VE : 0 . 68 ± 0 . 14 ( n = 115 ) ; USP28 in Ep : 1 . 00 ± 0 . 23 ( n = 280 ) ; USP28 in VE : 0 . 28 ± 0 . 00 ( n = 110 ) .

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

(C, D) Immunostaining for 53BP1 and USP28 on sagittal sections of WT embryos at E6.5 (C) or sagittal planes of whole-mount immunostaining at E5.5 (D). Quantification of the fluorescent signals in the epiblast (Ep) versus visceral endoderm (VE) are shown below. Four embryos per genotype were used for the quantifications. **** p < 0.0001, ***p<0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 50 μm. (C) 53BP1 in Ep: 1.00 ± 0.10 (n=225 cells); 53BP1 in VE: 0.37 ± 0.06 (n=105); USP28 in Ep: 1.00 ± 0.08 (n=235); USP28 in VE: 0.17 ± 0.05 (n=90). (D) 53BP1 in Ep: 1.00 ± 0.24 (n=225); 53BP1 in VE: 0.68 ± 0.14 (n=115); USP28 in Ep: 1.00 ± 0.23 (n=280); USP28 in VE: 0.28 ± 0.00 (n=110).


( A ) Immunostaining for centrosome marker γ - tubulin ( TUBG ) on Ctrl Sas - 4 − / − mESCs . bottom panels are magnifications of areas marked in top panels . Scale bars = 20 μm ( top ) 10 μm ( bottom ) .

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(A) Immunostaining for the centrosome marker γ-tubulin (TUBG) on Ctrl and Sas-4−/− primary mESCs. The bottom panels are magnifications of the areas marked in the top panels. Scale bars = 20 μm (top) and 10 μm (bottom).


( B ) Immunostaining for p53 on Ctrl Sas - 4 − / − mESCs in pluripotent partially differentiated ( diff . ) conditions . of p53 intensities was normalized to Ctrl ( Four independent experiments ) . * * p < 0 . 01 , * p < 0 . 05 ( two - student ' s T - , comparison of genotypes in " Pluripotent " condition was not significant using one - way ANOVA with Tukey ' s multiple comparisons ) . Bars represent mean ± s . d . Scale bar = 50 μm . Pluripotent Ctrl : 1 . 00 ± 0 . 02 ( n = 2030 ) ; pluripotent Sas - 4 - / - : 1 . 17 ± 0 . 10 ( n = 2170 ) ; partially diff . Ctrl : 0 . 62 ± 0 . 13 ( n = 2745 ) ; partially diff . Sas - 4 - / - : 1 . 12 ± 0 . 24 ( n = 2000 ) .

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(B) Immunostaining and quantification for p53 on Ctrl and Sas-4−/− primary mESCs in pluripotent and partially differentiated (diff.) conditions. The quantification of p53 fluorescence intensities was normalized to Ctrl (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test, the comparison of the genotypes in the "Pluripotent" condition was not significant using the one-way ANOVA with Tukey's multiple comparisons tests). Bars represent mean ± s.d. Scale bar = 50 μm. Pluripotent Ctrl: 1.00 ± 0.02 (n=2030 cells); pluripotent Sas-4-/-: 1.17 ± 0.10 (n=2170); partially diff. Ctrl: 0.62 ± 0.13 (n=2745); partially diff. Sas-4-/-: 1.12 ± 0.24 (n=2000).


( C , D ) Three - day growth curves of WT Sas - 4 − / − ( C ) or p53 − / − Sas - 4 − / − p53 − / − ( D ) mESCs in indicated conditions starting with 105 on Day 0 ( Four independent experiments ) . * * p < 0 . 01 , * p < 0 . 05 ( two - student ' s T - ) . Bars represent mean ± s . d . For details of measurements , see Materials Methods ( Tables 3 4 ) .

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(C, D) Three-day growth curves of WT and Sas-4−/− (C) or p53−/− and Sas-4−/− p53−/− (D) primary mESCs in the indicated conditions starting with 105 cells on Day 0 (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test). Bars represent mean ± s.d. For details of the measurements, see Materials and Methods (Tables 3 and 4).


( A ) Sagittal planes of whole - mount immunostaining for TUBG on WT mouse from E3 . 5 to E6 . 5 . insets are magnifications of selected centrosomes from dotted areas denoting inner mass ( E3 . 5 ) or epiblast ( E5 . 5 E6 . 5 ) . Bars represent mean ± s . d ( two - student ' s T - or one - way ANOVA with Tukey ' s multiple comparisons ) . Scale bars : E3 . 5 E5 . 5 = 15 μm ; E6 . 5 = 30 μm 3 μm ( insets ) . E3 . 5 : 88 ± 26 % ( n = 73 from 5 ) ; E5 . 5 : 92 ± 10 % ( n = 270 from 3 ) ; E6 . 5 : 79 ± 12 % ( n = 12 , 082 from 3 ) .

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(A) Sagittal planes of whole-mount immunostaining and quantification for TUBG on WT mouse embryos from E3.5 to E6.5. The insets are magnifications of selected centrosomes from the dotted areas denoting the inner cell mass (E3.5) or epiblast (E5.5 and E6.5). Bars represent mean ± s.d (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests). Scale bars: E3.5 and E5.5 = 15 μm; E6.5 = 30 μm and 3 μm (insets). E3.5: 88 ± 26% (n=73 cells from 5 embryos); E5.5: 92 ± 10% (n=270 from 3 embryos); E6.5: 79 ± 12% (n=12,082 from 3 embryos).


( B , C ) Representative gSTED super - resolution of immunostaining for TUBG either CEP164 ( B ) or ODF2 ( C ) . Whole - mount was used for CEP164 data as well as for E3 . 5 blastocysts for ODF2 , whereas on sections was used for ODF2 at E5 . 5 E6 . 5 . of of centrosomes ( TUBG ) with CEP164 or ODF2 are shown on right . * * * * p < 0 . 0001 , * * * p < 0 . 001 , * p < 0 . 05 ( two - student ' s T - or one - way ANOVA with Tukey ' s multiple comparisons ; ODF2 data between E5 . 5 E6 . 5 was not significant using latter ) . Bars represent mean ± s . d . Scale bar = 0 . 5 μm . CEP164 at E3 . 5 : 2 ± 2 % , ( n = 92 centrosomes from 4 ) ; E5 . 5 : 50 ± 2 % ( n = 249 from 3 ) ; E6 . 5 : 62 ± 0 % ( n = 856 from 3 ) . ODF2 at E3 . 5 : 20 ± 9 % ( n = 108 from 5 ) ; E5 . 5 : 51 ± 4 % ( n = 294 from 5 ) ; E6 . 5 : 60 ± 2 % ( n = 1 , 183 from 4 ) .

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

(B, C) Representative gSTED super-resolution images of immunostaining for TUBG and either CEP164 (B) or ODF2 (C). Whole-mount staining was used for the CEP164 data as well as for the E3.5 blastocysts for ODF2, whereas staining on sections was used for ODF2 at E5.5 and E6.5. Quantification of the percentage of centrosomes (TUBG) with CEP164 or ODF2 are shown on the right. **** p < 0.0001, *** p < 0.001, * p < 0.05 (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests; the ODF2 data between E5.5 and E6.5 was not significant using the latter test). Bars represent mean ± s.d. Scale bar = 0.5 μm. CEP164 at E3.5: 2 ± 2%, (n=92 centrosomes from 4 embryos); E5.5: 50 ± 2% (n=249 from 3 embryos); E6.5: 62 ± 0% (n=856 from 3 embryos). ODF2 at E3.5: 20 ± 9% (n=108 from 5 embryos); E5.5: 51 ± 4% (n=294 from 5 embryos); E6.5: 60 ± 2% (n=1,183 from 4 embryos).


( D ) Immunostaining of of TUBG PCNT signals from E5 . 5 E6 . 5 centrosomes in epiblast . separate channels for marked centriole are shown in insets . * * * p < 0 . 001 , * * p < 0 . 01 ( two - student ' s T - ) . Bars represent mean ± s . d . Scale bar = 2 μm . of PCNT at E5 . 5 : 0 . 22 ± 0 . 01 μm ( n = 103 centrosomes from 3 ) ; E6 . 5 : 0 . 31 ± 0 . 02 μm ( n = 342 from 3 ) . of TUBG at E5 . 5 : 0 . 15 ± 0 . 02 μm ( n = 108 from 4 ) ; E6 . 5 : 0 . 25 ± 0 . 01 μm ( n = 311 from 3 ) .

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(D) Immunostaining and quantification of the area of TUBG and PCNT signals from E5.5 and E6.5 centrosomes in epiblast cells. The separate channels for the marked centriole are shown in the insets. *** p < 0.001, ** p < 0.01 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 2 μm. Quantification of PCNT at E5.5: 0.22 ± 0.01 μm (n=103 centrosomes from 3 embryos); E6.5: 0.31 ± 0.02 μm (n=342 from 3 embryos). Quantification of TUBG at E5.5: 0.15 ± 0.02 μm (n=108 from 4 embryos); E6.5: 0.25 ± 0.01 μm (n=311 from 3 embryos).


( A ) Representative traces showing SQR - driven oxygen consumption in 3x106 control ( C ) five patientsfibroblasts ( P1 - 5 ) in absence ( solid traces ) or presence ( dashed traces ) of CoQ10 in culture medium . See Materials Methods for role of compounds used . ( B ) Relative rates of SQR - driven oxygen consumption are normalized to CIV respiration rates .

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(A) Representative traces showing SQR-driven oxygen consumption in 3x106 control (C) and five patientsfibroblasts (P1-5) in the absence (solid traces) or presence (dashed traces) of CoQ10 in the culture medium. See Materials and Methods for the role and concentration of the compounds used. (B) Relative rates of SQR-driven oxygen consumption are normalized to CIV respiration rates.


( A ) Representative western blot showing level of SQR , TST , ETHE1 SUOX in three control ( C1 - 3 ) four fibroblasts ( P1 - 4 ) . ( B - E ) Relative of normalized to Vinculin .

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(A) Representative western blot showing the level of SQR, TST, ETHE1 and SUOX proteins in three control (C1-3) and four patients fibroblasts (P1-4). (B-E) Relative levels of proteins normalized to Vinculin.


( A ) Representative western blots showing level of SQR , TST , ETHE1 SUOX in two controls ( C1 , C2 ) two fibroblasts ( P3 , P4 ) . ( B - E ) Relative of normalized to Vinculin .

(A) Representative western blots showing the level of SQR, TST, ETHE1 and SUOX proteins in two controls (C1, C2) and two patients fibroblasts (P3, P4). (B-E) Relative levels of proteins normalized to Vinculin.


( A ) Representative western blots showing SQR , TST , ETHE1 SUOX of two control fibroblasts treated with DMSO ( - ) or with DMSO + 4 - NB ( + ) . ( B ) of in fibroblasts treated with 4 - NB relative to control .

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(A) Representative western blots showing SQR, TST, ETHE1 and SUOX of two control fibroblasts treated with DMSO (-) or with DMSO+ 4-NB (+). (B) Quantification of proteins levels in fibroblasts treated with 4-NB relative to control.


( C ) Representative western blots showing ADCK3 , SQR , TST , ETHE1 SUOX in two control ( EV ) six ADCK3 - depleted clones ( ADCK3 RNAi ) . ( D ) of in ADCK3 - depleted clones ( ADCK3 RNAi ) relative to controls ( EV ) .

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(C) Representative western blots showing ADCK3, SQR, TST, ETHE1 and SUOX in two control (EV) and six ADCK3-depleted clones (ADCK3 RNAi). (D) Quantification of proteins levels in ADCK3-depleted clones (ADCK3 RNAi) relative to controls (EV).


Distribution of peptides containing sulfhydrated cysteine residues relative to their H / L ratios as determined by BTA of extracts isolated from C1 P1 ( indicated in A ) C2 P4 fibroblasts ( B ) . Values of H / L ratios are plotted against number of identified peptides . gray marks H / L > 2 , consisting of cysteine - containing peptides in from lines that exhibited higher reactivity with H2S compared with respective control . Indicated on X axes are representative sensitive cysteine peptides , which are common between ones found in RedoxDB database , by BTA .

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Distribution of peptides containing sulfhydrated cysteine residues relative to their H/L ratios as determined by the BTA analysis of cell extracts isolated from C1 and P1 patient cells (indicated in A) and C2 and P4 fibroblasts (B). Values of H/L ratios are plotted against the number of identified peptides. The gray line marks the H/L ratio >2, consisting of cysteine-containing peptides in proteins from patient cell lines that exhibited higher reactivity with H2S compared with the respective control. Indicated on the X axes are representative sensitive cysteine peptides, which are common between the ones found in the RedoxDB database, and by the BTA assay.


( A ) Representative western blots showing of SQR , TST , ETHE1 SUOX in kidney of three wild - type ( WT ) three mutant ( Mut ) mice . ( B - E ) Relative of normalized to Vinculin .

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(A) Representative western blots showing the levels of SQR, TST, ETHE1 and SUOX protein in the kidney of three wild-type (WT) and three mutant (Mut) mice. (B-E) Relative levels of proteins normalized to Vinculin.


( A ) Representative western blots showing of SQR , TST , ETHE1 SUOX from brain of 5 wild - type ( WT ) five mutant ( Mut ) mice ( B - E ) Relative of normalized to Vinculin .

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(A) Representative western blots showing the levels of SQR, TST, ETHE1 and SUOX protein from the brain of 5 wild-type (WT) and five mutant (Mut) mice (B-E) Relative levels of proteins normalized to Vinculin.


( A ) Total GSH amount normalized to from kidney ( B ) brain of wild - type ( WT ) mutant ( Mut ) mice . Error bars represent SDs of at least 3 per group .

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(A) Total GSH amount normalized to protein from kidney and (B) brain of wild-type (WT) and mutant (Mut) mice. Error bars represent SDs of at least 3 animals per group.


( C ) Thiosulfate in urine ( D ) plasma normalized to amount . Error bars represent SDs of 9 per group ( urine ) 6 per group ( plasma ) .

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(C) Thiosulfate levels in urine and (D) plasma normalized to protein amount. Error bars represent SDs of 9 animals per group (urine) and 6 animals per group (plasma).


( E ) H2S amount normalized to tissue from kidney ( F ) brain . Error bars represent SDs of 7 / 8 per group .

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(E) H2S amount normalized to tissue from kidney and (F) brain. Error bars represent SDs of 7/8 animals per group.


( G ) Acylcarnitine C4 , C5 C6 from plasma , normalized to volume . Error bars represent SDs of 5 wild - type 3 mutants .

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(G) Acylcarnitine C4, C5 and C6 levels from plasma, normalized to volume. Error bars represent SDs of 5 wild-type and 3 mutants.


B . Table showing location , number of inserted or deleted residues motility phenotype of 18 stalk mutants . classification are on three technical repetitions . C . Example kymographs for ' Directional - WT like ' , ' Diffusive like ' , ' No movement ' , ' Transient binding ' . Magnified for ' Diffusive like ' motion shows run of a single molecule .

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B. Table showing location, number of inserted or deleted residues and motility phenotype of all 18 stalk mutants. Quantification and classification are based on three technical repetitions. C. Example kymographs for 'Directional - WT like', 'Diffusive like', 'No movement', and 'Transient binding'. Magnified area for 'Diffusive like' motion shows run of a single molecule.


D . Table showing type of movement found for wild - type , mutant 2 , mutant 5 , mutant 13 in a modified single - molecule with without ATP . Classification of type of movement is on two repetitions of different dynein preparations .

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D. Table showing the type of movement found for wild-type, mutant 2, mutant 5, and mutant 13 in a modified single-molecule assay with and without ATP. Classification of type of movement is based on two repetitions of different dynein preparations.


A - D Microtubule affinity measured by a cosedimentation in apo state ( full ) in presence of ATP ( dashed ) , AMPPNP ( dotted ) for wild - type ( A ) , mutant 2 ( B ) , mutant 5 ( C ) , mutant 13 ( D ) .

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A-D Microtubule affinity measured by a cosedimentation assay in the apo state (full line) and in the presence of ATP (dashed line), and AMPPNP (dotted line) for wild-type (A), mutant 2 (B), mutant 5 (C), and mutant 13 (D).


E . Microtubule stimulated ATPase activity of wild - type ( grey ) , mutant 2 ( red ) , mutant 5 ( orange ) , mutant 13 ( purple ) . F . Bar plot of basal ATPase activity of wild - type ( grey ) , mutant 2 ( red ) , mutant 5 ( orange ) , mutant 13 ( purple ) . Data information : Error bars show standard deviation of three repetitions of different dynein preparations .

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E. Microtubule stimulated ATPase activity of wild-type (grey), mutant 2 (red), mutant 5 (orange), and mutant 13 (purple). F. Bar plot of basal ATPase activity of wild-type (grey), mutant 2 (red), mutant 5 (orange), and mutant 13 (purple). Data information: Error bars show standard deviation of three repetitions of different dynein preparations.


A captured by BGL3 RMRP antisense purifications . with an absolute log2 ( fold change ) > 1 - log10 ( p - value ) > 1 . 3 ( compared with control purification ) were considered as enriched by BGL3 specifically .

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A Proteins captured by BGL3 and RMRP antisense purifications. Proteins with an absolute log2 (fold change) > 1 and -log10 (p-value) > 1.3 (compared with control purification) were considered as proteins enriched by BGL3 specifically.


C & D pull - down for BGL3 - BARD1 / PARP1 interactions before after irradiation . Biotinylated in vitro - transcribed LncRNA - BGL3 sense or antisense transcripts were incubated with HEK - 293T lysates with or without ionizing radiation ( 10 Gy ) with 1 hr recovery for in vitro streptavidin pull - down assays , followed by Western blots using indicated antibodies .

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C&D RNA pull-down assay for BGL3-BARD1/PARP1 interactions before and after irradiation. Biotinylated in vitro-transcribed LncRNA-BGL3 sense or antisense transcripts were incubated with HEK-293T cell lysates with or without ionizing radiation (10 Gy) with 1 hr recovery for in vitro streptavidin RNA pull-down assays, followed by Western blots using the indicated antibodies.


HA - tagged full - or deletion mutants of BARD1 were transfected into 293T . 48 hr later , BGL3 pull - down were performed , arrowheads indicate bands for HA - tagged full - or deletion mutants of

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HA-tagged full-length or deletion mutants of BARD1 were transfected into 293T cells. 48 hr later, BGL3 RNA pull-down assay were performed, and arrowheads indicate the bands for HA-tagged full-length or deletion mutants of BARD1


HA - tagged full - or deletion mutants of PARP1 ( H ) were transfected into 293T . 48 hr later , BGL3 pull - down were performed ,

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HA-tagged full-length or deletion mutants of PARP1(H) were transfected into 293T cells. 48 hr later, BGL3 RNA pull-down assay were performed,


A BGL3 is recruited to damage sites . U2OS were subjected to micro - irradiation to generate DSBs in a pattern . relocation kinetics of BGL3 to DSBs was monitored by in situ hybridization ( FISH ) in a time course as indicated . BGL3 intensities at were normalized into a numerical value using Nikon NIS - Elements AR software ( version 4 . 40 . 00 ) . Data are presented as mean ± SD of four biological replicates .

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A BGL3 is recruited to DNA damage sites. U2OS cells were subjected to laser micro-irradiation to generate DSBs in a line pattern. The relocation kinetics of BGL3 to DSBs was monitored by RNA fluorescence in situ hybridization (FISH) in a time course as indicated. BGL3 intensities at the laser line were normalized into a numerical value using Nikon NIS-Elements AR software (version 4.40.00). Data are presented as mean ±SD of four biological replicates.


B Chromosome aberrations induced by BGL3 depletion . Wild - type or BGL3 depleted BJ - 5ta were used in metaphase spread for spontaneous breaks . Representative image ( left panel ) of containing at least one break ( right panel ) . Data are presented as mean ± SD of three biological replicates . Two - Student ' s t , * * P < 0 . 01 .

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B Chromosome aberrations induced by BGL3 depletion. Wild-type or BGL3 depleted BJ-5ta cells were used in the metaphase spread analysis for spontaneous DNA breaks. Representative image (left panel) and the percentage of cells containing at least one DNA break (right panel). Data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, ** P<0.01.


C MCF - 7 were treated as indicated , response to ionizing radiation were analyzed by colony formation assays ( left panel ) . Data are presented as mean ± of four independent experiments , analyzed by two - way of variance ( ANOVA ) , * * P < 0 . 01 . Knock - down efficiency of BGL3 siRNA was examined by qRT - PCR normalized to β - Actin ( middle panel ) , data are presented as mean ± SD of three biological replicates . Two - Student ' s t , * * P < 0 . 01 . Knock - down efficiency of CtIP siRNA was examined by blotting ( right panel ) .

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C MCF-7 cells were treated as indicated, and cell response to ionizing radiation were analyzed by colony formation assays (left panel). Data are presented as mean ± SEM of four independent experiments, analyzed by two-way analysis of variance (ANOVA), **P < 0.01. Knock-down efficiency of BGL3 siRNA was examined by qRT-PCR and normalized to β-Actin (middle panel), data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, **P < 0.01. Knock-down efficiency of CtIP siRNA was examined by blotting (right panel).


D MCF - 7 were transfected with indicated siRNAs . sensitivity to camptothecin ( CPT ) , hydroxyurea ( HU ) , etoposide ( ETO ) , or ionizing radiation was determined by MTS assays . Data are presented as mean ± SD of three biological replicates . Two - Student ' s t , * P < 0 . 05 , * * P < 0 . 01 , NS : no significant difference .

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D MCF-7 cells were transfected with the indicated siRNAs. Cell sensitivity to camptothecin (CPT), hydroxyurea (HU), etoposide (ETO), or ionizing radiation was determined by MTS assays. Data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, *P < 0.05, **P < 0.01, NS: no significant difference.


E BGL3 deficiency inhibits damage repair . of γ - H2AX foci at indicated times after irradiation ( 2Gy ) are presented . Data shown are results of three independent experiments ( 100 for each experiment ) , presented as mean ± SD , two - Student ' s t , * P < 0 . 05 , * * P < 0 . 01 , NS : no significant difference .

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E BGL3 deficiency inhibits DNA damage repair. Quantification of γ-H2AX foci at indicated times after irradiation (2Gy) are presented. Data shown are results of three independent experiments (100 cells for each experiment), presented as mean ± SD, two-tailed Student's t test, *P < 0.05, **P < 0.01, NS: no significant difference.


A & B HEK293 integrated with HR or NHEJ reporter were transfected with indicated siRNAs subjected to HR ( A ) or NHEJ ( B ) as described in Methods . Data presented as mean ± SD of four biological replicates , positive percentages as compared with control group . Two - Student ' s t , * * P < 0 . 01 . NS : no significant difference .

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A&B HEK293 cells integrated with HR or NHEJ reporter were transfected with the indicated siRNAs and subjected to the HR (A) or NHEJ assay (B) as described in Methods. Data presented as mean ±SD of four biological replicates, and positive cell percentages as compared with control group. Two-tailed Student's t test, **P < 0.01. NS: no significant difference.


C - F Wild - type ( CTRL ) or BGL3 knockdown U2OS were subjected to micro - irradiation ; 1 hr later , were fixed processed for immunostaining with indicated antibodies . Representative of BRCA1 ( C ) , BARD1 ( D ) , RAD51 ( E ) , RPA70 ( F ) accumulation at sites of - induced damage are shown .

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C-F Wild-type (CTRL) or BGL3 knockdown U2OS cells were subjected to micro-irradiation; 1 hr later, cells were fixed and processed for immunostaining with indicated antibodies. Representative images of BRCA1 (C), BARD1 (D), RAD51 (E), and RPA70 (F) accumulation at sites of laser-induced DNA damage are shown.


Parental or BGL3 - deficient U2OS were subjected to micro - irradiation to generate DSBs in a pattern . DDR factor recruitment were examined 2 hr later . Data shown are average of three independent experiments , 100 were counted for each experiment . Data are presented as mean ± SD , analyzed by two - Student ' s t , * * P < 0 . 01 .

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Parental or BGL3-deficient U2OS cells were subjected to laser micro-irradiation to generate DSBs in a line pattern. DDR factor recruitment were examined 2 hr later. Data shown are the average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, **P < 0.01.


U2OS were transfected with indicated siRNAs , 48 hr later , HR efficiency were determined using reporter , as described in Methods . Data shown are average of three independent experiments . Data are presented as mean ± SD , analyzed by two - Student ' s t , NS : no significant difference .

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U2OS cells were transfected with the indicated siRNAs, and 48 hr later, HR efficiency were determined using the reporter assay, as described in the Methods. Data shown are the average of three independent experiments. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, NS: no significant difference.


E U2OS were transfected with indicated siRNA , RAD51 accumulation at sites of - induced damage were examined . Data shown are average of three independent experiments , 100 were counted for each experiment . Data are presented as mean ± SD , analyzed by two - Student ' s t , NS : no significant difference . Knock - down efficiency of BARD1 siRNA was examined by blotting ( bottom left panel ) , knock - down efficiency of BGL3 siRNA was examined by qRT - PCR normalized to β - actin ( bottom right panel ) , data are presented as mean ± SD of three biological replicates . Two - Student ' s t , * * P < 0 . 01 .

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E U2OS cells were transfected with the indicated siRNA, and RAD51 accumulation at sites of laser-induced DNA damage were examined. Data shown are the average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, NS: no significant difference. Knock-down efficiency of BARD1 siRNA was examined by blotting (bottom left panel), and knock-down efficiency of BGL3 siRNA was examined by qRT-PCR and normalized to β-actin (bottom right panel), data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, **P < 0.01.


A BGL3 promotes BARD1 - HP1γ interactions . 293T expressing indicated siRNAs were irradiated ( 10 Gy ) , BARD1 - HP1γ interactions examined at indicated time points .

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A BGL3 promotes BARD1-HP1γ interactions. 293T cells expressing the indicated siRNAs were irradiated (10 Gy), and BARD1-HP1γ interactions examined at the indicated time points.


B BGL3 promotes RAP80 binding to K63 ubiquitin chain in . 293T expressing indicated siRNAs plasmids were irradiated ( 10 Gy ) . lysates were immunoprecipitated with HA beads subjected to immunoblot with indicated antibodies .

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B BGL3 promotes RAP80 binding to K63 ubiquitin chain in cells. 293T cells expressing the indicated siRNAs and plasmids were irradiated (10 Gy). Cell lysates were immunoprecipitated with HA beads and subjected to immunoblot with the indicated antibodies.


C BGL3 promotes BARD1 - HP1γ ( up panel ) BARD1 - RAD51 ( middle panel ) interactions in vitro . GST - HP1γ , GST - RAD51 , FLAG - BARD1 were overexpressed purified from as indicated in Methods . FLAG - BARD1 was divided into two equal parts , one part was used for BARD1 - HP1γ interaction ( up panel ) , another part was used for BARD1 - RAD51 interaction ( middle panel ) . BARD1 - HP1γ ( up panel ) BARD1 - RAD51 ( middle panel ) interactions with or without BGL3 were respectively detected by GST pull - down assays , here GST was used as a pull - down control .

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C BGL3 promotes BARD1-HP1γ (up panel) and BARD1-RAD51 (middle panel) interactions in vitro. GST-HP1γ, GST-RAD51, and FLAG-BARD1 were overexpressed and purified from cells as indicated in Methods. FLAG-BARD1 was divided into two equal parts, one part was used for BARD1-HP1γ interaction analysis (up panel), and another part was used for BARD1-RAD51 interaction analysis (middle panel). BARD1-HP1γ (up panel) and BARD1-RAD51 (middle panel) interactions with or without BGL3 were respectively detected by GST pull-down assays, here GST was used as a pull-down control.


A PARP1 inhibitor treatment abolished BGL3 recruitment to DSBs . U2OS were treated with DMSO , PARP inhibitor ( olaparib ) , ATM inhibitor ( KU - 55933 ) , ATR inhibitor ( VE - 821 ) or caffeine . BGL3 recruitment to damage sites was assessed by - FISH assays . B of positive . For each group , 100 randomly selected were counted . Data are presented as mean ± SD of three biological replicates . Two - Student ' s t , * * P < 0 . 01 , N : no significant difference .

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

A PARP1 inhibitor treatment abolished BGL3 recruitment to DSBs. U2OS cells were treated with DMSO, PARP inhibitor (olaparib), ATM inhibitor (KU-55933), ATR inhibitor (VE-821) or caffeine. BGL3 recruitment to DNA damage sites was assessed by RNA-FISH assays. B Quantification of the positive cells. For each group, 100 randomly selected cells were counted. Data are presented as mean ±SD of three biological replicates. Two-tailed Student's t test, **P < 0.01, N: no significant difference.


C PARP inhibitor mainly affected BGL3 recruitment at an early time point . U2OS were treated with DMSO or olaparib , BGL3 recruitment was examined following micro - irradiation at indicated time point . Data presented are average of three independent experiments , 100 were counted for each experiment . Two - Student ' s t , * * P < 0 . 01 , N : no significant difference .

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

C PARP inhibitor mainly affected BGL3 recruitment at an early time point. U2OS cells were treated with DMSO or olaparib, and BGL3 recruitment was examined following laser micro-irradiation at indicated time point. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference.


D U2OS were transfected with indicated siRNAs , BGL3 recruitment to DSBs were monitored at indicated time point by FISH . Data presented are average of three independent experiments , 100 were counted for each experiment . Two - Student ' s t , * * P < 0 . 01 , N : no significant difference .

O O O O 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

D U2OS cells were transfected with the indicated siRNAs, and BGL3 recruitment to DSBs were monitored at the indicated time point by RNA FISH. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference.