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 .

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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 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 .

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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 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 .

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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 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 .

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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.


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 .

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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 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 .

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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 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 .

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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 , 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 ) .

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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 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 ) .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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 , 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 ) .

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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 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 ) .

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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 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 .

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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 ( 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 .

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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 , 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 .

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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 , 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 .

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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 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 .

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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 ) 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 ) .

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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 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 ) .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 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 .

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

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 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 .

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

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 , 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 .

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

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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

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 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 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 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 .

O O B-MEASURED_VAR 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 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 and eControl dams ( mean ± SEM , Student ' s t test ; 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 , 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 .

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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 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 .

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 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 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 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 .

O 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 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

(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 ) 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 ) .

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

O O O O O O O O O O O O B-MEASURED_VAR 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-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 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 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 ) .

O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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(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 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 ) .

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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 ) 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 .

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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 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 .

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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 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 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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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) 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 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 .

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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 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 ) .

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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 and ( F ) brain . Error bars represent SDs of 7 / 8 animals 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 and C6 levels from plasma , normalized to volume . Error bars represent SDs of 5 wild - type and 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 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 .

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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 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 .

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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 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 ) .

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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 ) , 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 .

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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 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 .

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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 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 .

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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 - 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

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

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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 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 .

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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 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 .

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

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

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 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 .

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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 ) 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 .

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

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 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 .

O B-CONTROLLED_VAR O O O B-MEASURED_VAR O 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 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

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 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 .

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

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 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 .

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

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.


B . Total quantified proteome depth in tissues ( n = 18 ) , primary cells ( n = 12 ) , immortalized cell lines ( n = 4 ) and all samples ( n = 34 ) . In all cases , n means biological replicates unless otherwise indicated . The upper - and lower panel shows the number of quantified protein groups and peptides , respectively . C . Dynamic range of the different proteomes based on median LFQ intensity ordered by abundance rank ( Liver : bulk liver biopsy , HepA : hepatic artery , PorV : portal vein , Cell lines : mixture of human liver - derived immortalized cell lines ) . Number of biological replicates is same as Panel ( A ) .

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B. Total quantified proteome depth in tissues (n=18), primary cells (n=12), immortalized cell lines (n=4) and all samples (n=34). In all cases, n means biological replicates unless otherwise indicated. The upper- and lower panel shows the number of quantified protein groups and peptides, respectively. C. Dynamic range of the different proteomes based on median LFQ intensity ordered by abundance rank (Liver: bulk liver biopsy, HepA: hepatic artery, PorV: portal vein, Cell lines: mixture of human liver-derived immortalized cell lines). Number of biological replicates is same as Panel (A).


B . Cumulative protein abundance of liver biopsy , haptic artery and portal vein as a function of protein rank , with the total of the top ten abundant proteins and number of proteins that comprise four quartiles indicated .

O O O O O O O O O O O O O O O O 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. Cumulative protein abundance of liver biopsy, haptic artery and portal vein as a function of protein rank, with the total of the top ten abundant proteins and number of proteins that comprise four quartiles indicated.


D . Cumulative protein abundance of liver cell types as a function of protein rank , indicating the total of the top ten abundant proteins and number of proteins that comprise the four quartiles .

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D. Cumulative protein abundance of liver cell types as a function of protein rank, indicating the total of the top ten abundant proteins and number of proteins that comprise the four quartiles.


F . The bar plot shows the contribution of each organelle to total cellular protein mass , also accounting for unassigned proteins . Percentage of cytosol , mitochondria , nucleoli , plasma membrane and actin filaments is indicated .

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

F. The bar plot shows the contribution of each organelle to total cellular protein mass, also accounting for unassigned proteins. Percentage of cytosol, mitochondria, nucleoli, plasma membrane and actin filaments is indicated.


A . Total protein copy number of the oxidative phosphorylation complex I - V in bulk liver tissue ( n = 6 independent biological replicates ) and primary cell types ( isolated primary cells from n = 3 individuals for each cell type ) . Values are presented as mean ± s . d . B . Protein copy number for members of the oxidative phosphorylation complex I - V in hepatocytes and Kupffer cells with Pearson correlation coefficient indicated .

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A. Total protein copy number of the oxidative phosphorylation complex I-V in bulk liver tissue (n=6 independent biological replicates) and primary cell types (isolated primary cells from n=3 individuals for each cell type). Values are presented as mean ± s.d. B. Protein copy number for members of the oxidative phosphorylation complex I-V in hepatocytes and Kupffer cells with Pearson correlation coefficient indicated.


D . Top ten uniquely quantified proteins per cell type with LFQ intensities [ Log10 ] and copy numbers [ Log10 ] .

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

D. Top ten uniquely quantified proteins per cell type with LFQ intensities [Log10] and copy numbers [Log10].


A . Unsupervised hierarchical clustering of proteins significantly differentially abundant across cell types , with columns showing three biological replicates ( n = 3 ) of four cell types and rows significant proteins . Significance was calculated by one - way ANOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) . Frames and numbers indicate four clusters of proteins highly enriched in each cell type .

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A. Unsupervised hierarchical clustering of proteins significantly differentially abundant across cell types, with columns showing three biological replicates (n=3) of four cell types and rows significant proteins. Significance was calculated by one-way ANOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05). Frames and numbers indicate four clusters of proteins highly enriched in each cell type.


A . Total quantified proteins at day one , three and seven upon primary cell culture in hepatocytes ( hHEP ) , hepatic stellate cells ( hHSC ) and liver sinusoidal endothelial cells ( LSEC ) . In all cases , number of biological replicates is 3 . Values are presented as mean ± s . d . No additional replications of the experiment was done in laboratory .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Total quantified proteins at day one, three and seven upon primary cell culture in hepatocytes (hHEP), hepatic stellate cells (hHSC) and liver sinusoidal endothelial cells (LSEC). In all cases, number of biological replicates is 3. Values are presented as mean ± s.d. No additional replications of the experiment was done in laboratory.


F . Protein expression patterns over the course of primary hHSC culture ( n = 3 ) . Values are presented as mean ± s . d . Significance was calculated by ANOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) with a significance level of * * P < 0 . 01 , and * * * P < 0 . 001 .

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F. Protein expression patterns over the course of primary hHSC culture (n=3). Values are presented as mean ± s.d. Significance was calculated by ANOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05) with a significance level of **P < 0.01, and ***P < 0.001.


A . Hierarchical clustering of proteins significantly differentially abundant between NASH ( n = 20 ) , cirrhosis ( n = 10 ) and control groups ( n = 15 ) . Significance was calculated by ANCOVA , followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) . Two major clusters of proteins were identified with Cluster 1 mainly upregulated in cirrhosis compared to NASH and controls and Cluster 2 downregulated . No additional replications of the experiment was done in laboratory .

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A. Hierarchical clustering of proteins significantly differentially abundant between NASH (n=20), cirrhosis (n=10) and control groups (n=15). Significance was calculated by ANCOVA, followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05). Two major clusters of proteins were identified with Cluster 1 mainly upregulated in cirrhosis compared to NASH and controls and Cluster 2 downregulated. No additional replications of the experiment was done in laboratory.


D . Box ‐ and ‐ whisker plot showing the distribution of log2 ‐ intensity values of statistical significantly regulated proteins across three groups . Number of independent biological replicates is n = 15 , 20 and 10 for the control , NASH and cirrhosis group , respectively . The black line in the middle of the box is the median , the top and the bottom of the box represent the upper and lower quartile values of the data and the whiskers represent the upper and lower limits for considering outliers ( Q3 + 1 . 5 * IQR , Q1 ‐ 1 . 5 * IQR ) where IQR is the interquartile range ( Q3 - Q1 ) . Significance was defined by ANCOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) with a significance level of * * * P < 0 . 001 . E . Heatmap of CYP 450 family members that are statistically significantly abundant between three groups . Data was presented as mean protein abundance followed by Z - score normalization across . Number of replicates is same as Panel ( C ) . F . Extracellular matrix ( ECM ) remodeling in liver cirrhosis . Upper and lower panel shows the abundance rank of proteins involved in ECM organization in the control ( upper ) and cirrhosis ( lower ) groups , highlighting top shifted ECM components in the cirrhosis group as compared with the control group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Box‐and‐whisker plot showing the distribution of log2‐intensity values of statistical significantly regulated proteins across three groups. Number of independent biological replicates is n=15, 20 and 10 for the control, NASH and cirrhosis group, respectively. The black line in the middle of the box is the median, the top and the bottom of the box represent the upper and lower quartile values of the data and the whiskers represent the upper and lower limits for considering outliers (Q3+1.5*IQR, Q1‐1.5*IQR) where IQR is the interquartile range (Q3-Q1). Significance was defined by ANCOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05) with a significance level of ***P < 0.001. E. Heatmap of CYP 450 family members that are statistically significantly abundant between three groups. Data was presented as mean protein abundance followed by Z-score normalization across. Number of replicates is same as Panel (C). F. Extracellular matrix (ECM) remodeling in liver cirrhosis. Upper and lower panel shows the abundance rank of proteins involved in ECM organization in the control (upper) and cirrhosis (lower) groups, highlighting top shifted ECM components in the cirrhosis group as compared with the control group.


Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical plane . The apical MTs were associated with both LUZP1 and TJs . Scale bar , 10 μm ( low magnification ) and 1 μm ( high magnification ) . Representative immunoelectron micrographs of Eph4 cells . Immunogold particles for LUZP1 preferentially accumulated at the level of TJs that are positive for cingulin . MV , microvilli ; AJ , adherens junction , DS , desmosome . Scale bars , 200 nm .

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Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical plane. The apical MTs were associated with both LUZP1 and TJs. Scale bar, 10 μm (low magnification) and 1 μm (high magnification).   Representative immunoelectron micrographs of Eph4 cells. Immunogold particles for LUZP1 preferentially accumulated at the level of TJs that are positive for cingulin. MV, microvilli; AJ, adherens junction, DS, desmosome. Scale bars, 200 nm.


Representative confocal micrographs of co - cultures of wild - type ( WT ) and ZO - 1 / - 2 double knockout ( DKO ) Eph4 cells . ZO - 1 / - 2 DKO cells were marked by asterisks ( * ) . The LUZP1 junctional localization was apparently disrupted in TJ - deficient ZO - 1 / - 2 DKO cells . Scale bar , 10 μm . Representative confocal micrographs of transient E - cadherin knockout ( KO ) in WT Eph4 cells . E - cadherin KO cells were marked by asterisks ( * ) . The LUZP1 junctional localization seemed not to change between AJ - deficient E - cadherin KO and WT cells . Scale bar , 10 μm .

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

Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ZO-1/-2 DKO cells were marked by asterisks (*). The LUZP1 junctional localization was apparently disrupted in TJ-deficient ZO-1/-2 DKO cells. Scale bar, 10 μm.   Representative confocal micrographs of transient E-cadherin knockout (KO) in WT Eph4 cells. E-cadherin KO cells were marked by asterisks (*). The LUZP1 junctional localization seemed not to change between AJ-deficient E-cadherin KO and WT cells. Scale bar, 10 μm.


Bar plots with dot density plots showing that ppMLC levels within CRs were significantly reduced in LUZP1 KO cells compared to those in WT cells [ 19 . 19 ± 10 . 02 arbitrary units ( a . u . ) ( WT ) vs . 2 . 92 ± 1 . 81 a . u . ( LUZP1 KO ) ] . n = 3 . * * p < 0 . 01 ( Mann - Whitney U test ) . Bars and error bars represent the mean ± SD .

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Bar plots with dot density plots showing that ppMLC levels within CRs were significantly reduced in LUZP1 KO cells compared to those in WT cells [19.19 ± 10.02 arbitrary units (a.u.) (WT) vs. 2.92 ± 1.81 a.u. (LUZP1 KO)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± SD.


Representative confocal micrographs of co - cultures of wild - type ( WT ) and ZO - 1 / - 2 double knockout ( DKO ) Eph4 cells . ppMLC levels within CRs were clearly reduced in ZO - 1 / - 2 DKO cells compared to those in WT cells . Scale bar , 10 μm . Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of ZO - 1 / - 2 DKO cells [ 119 . 62 ± 31 . 62 μm2 ( WT ) vs . 199 . 96 ± 94 . 22 μm2 ( LUZP1 KO ) ] ( See also Fig 1I ) . n = 3 . * * p < 0 . 01 ( unpaired t test ) . Bars and error bars represent the mean ± SD .

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 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 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 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

Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ppMLC levels within CRs were clearly reduced in ZO-1/-2 DKO cells compared to those in WT cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of ZO-1/-2 DKO cells [119.62 ± 31.62 μm2 (WT) vs. 199.96 ± 94.22 μm2 (LUZP1 KO)] (See also Fig 1I). n = 3. **p < 0.01 (unpaired t test). Bars and error bars represent the mean ± SD.