Representative confocal images of STX in the Cortex , Hippocampus , and Cerebellum brain section derived from 7 . 5 months - old mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen ( Tamox ) . Quantification of confocal images , the plot shows the quantification of the STX average spot area normalized for the number of Hoechst positive cells . ( * * * vs WT , ° ° ° / ° ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * * / oo : P ≤ 0 . 001 , * * * / ooo : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological replicas ) . Scale bars : 60 µm .

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

Representative confocal images of STX in the Cortex, Hippocampus, and Cerebellum brain section derived from 7.5 months-old mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen (Tamox). Quantification of confocal images, the plot shows the quantification of the STX average spot area normalized for the number of Hoechst positive cells. (*** vs WT, °°°/°° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, **/oo: P ≤ 0.001, ***/ooo: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 60 µm.


Representative confocal images of SCMAS in the Cortex , Hippocampus , and Cerebellum brain section derived from mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen . Quantification of confocal images , the plot shows the quantification of the SCMAS average spot area normalized for the number of Hoechst positive cells . ( * * * vs WT , ° ° ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological ) . Scale bars : 60 µm .

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

Representative confocal images of SCMAS in the Cortex, Hippocampus, and Cerebellum brain section derived from mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen. Quantification of confocal images, the plot shows the quantification of the SCMAS average spot area normalized for the number of Hoechst positive cells. (*** vs WT, °°° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological). Scale bars: 60 µm.


( A - B ) Representative confocal images and quantification of IBA - 1 in the Cortex , Hippocampus , and Cerebellum brain section derived from WT or CLN7Δex2 mice injected with the vehicle or Tamoxifen . ( * * * / * * / * vs WT , ° ° ° / ° ° / ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * / o : P ≤ 0 . 01 , * * / oo : P ≤ 0 . 001 , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological replicas ) . Scale bars : 50 µm .

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

(A-B) Representative confocal images and quantification of IBA-1 in the Cortex, Hippocampus, and Cerebellum brain section derived from WT or CLN7Δex2 mice injected with the vehicle or Tamoxifen. (***/**/* vs WT, °°°/°°/° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, */o: P ≤ 0.01, **/oo: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 50 µm.


( D ) Representative Images of hindlimb clasping test in mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen .

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

(D) Representative Images of hindlimb clasping test in mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen.


A HeLa - H2B / tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine ( TT ) block . After release from the block , mitotic cells were counted by fluorescence microscopy ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

A HeLa-H2B/tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine (TT) block. After release from the block, mitotic cells were counted by fluorescence microscopy (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


B Time from S - to M - phase was measured in si - RNA transfected HeLa - H2B / tub cells by time - lapse microscopy following release from TT block ( 4 repeat experiments ; all measurements depicted ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

B Time from S- to M-phase was measured in si-RNA transfected HeLa-H2B/tub cells by time-lapse microscopy following release from TT block (4 repeat experiments; all measurements depicted). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


Western blot analysis of unsynchronized HeLa - H2B / tub cells was carried out to monitor expression of p ( S10 ) - H3 , total H3 and TIAR in control and TIAR - depleted cells ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

Western blot analysis of unsynchronized HeLa-H2B/tub cells was carried out to monitor expression of p(S10)-H3, total H3 and TIAR in control and TIAR-depleted cells (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


D HeLa cells were transfected with control or TIAR si - RNAs , alone or together with Cdc25B siRNA . 72 hours after transfection , p ( S10 ) - H3 - positive cells were quantified by flow cytometry ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

D HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA. 72 hours after transfection, p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


HeLadox - YFP - TIARr cells were transfected with control or TIAR siRNAs , and 24h hours later cell were cultured in the abscence or presence of 1 μg / ml doxocycline for 48 hours . The expression of p ( S10 ) - H3 , total H3 and TIAR was measured by Westen blot analysis . The graph shows the quantification of p - H3 levels normalized to total H3 ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

HeLadox-YFP-TIARr cells were transfected with control or TIAR siRNAs, and 24h hours later cell were cultured in the abscence or presence of 1 μg/ml doxocycline for 48 hours. The expression of p(S10)-H3, total H3 and TIAR was measured by Westen blot analysis. The graph shows the quantification of p-H3 levels normalized to total H3 (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


F HeLadox - YFP - TIARr - RRM123m cells were analyzed as in panel ( E ) ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

F HeLadox-YFP-TIARr-RRM123m cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


G HeLadox - YFP - TIARr - dQRD cells were analyzed as in panel ( E ) ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

G HeLadox-YFP-TIARr-dQRD cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


A Following transfection of HeLa cells with control or TIAR siRNAs for 72 hours , mitotic cells were analyzed by IF microscopy and scored for chromatin ( mean ± SE , n = 3 ) . B HeLa cells were analyzed as in ( A ) and scored for spindle defects ( mean ± SE , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

A Following transfection of HeLa cells with control or TIAR siRNAs for 72 hours, mitotic cells were analyzed by IF microscopy and scored for chromatin (mean ± SE, n = 3). B HeLa cells were analyzed as in (A) and scored for spindle defects (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


C HeLa cells were transfected with control or TIAR siRNAs for 4 days , and subjected to colcemid for 2 hours prior to preparation of metaphase spreads . Chromosomes were stained with Hoechst ( red ) and anti - CENP - A antibody ( green ) ; the yellow arrowhead marks a chromosome break .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with control or TIAR siRNAs for 4 days, and subjected to colcemid for 2 hours prior to preparation of metaphase spreads. Chromosomes were stained with Hoechst (red) and anti-CENP-A antibody (green); the yellow arrowhead marks a chromosome break.


D Metaphase spreads were prepared as in ( C ) , and the frequency of cells with chromosomal breaks was quantified ( mean ± SD , n = 3 independent experiments , approximately 30 metaphase spreads were assessed per experiment and condition ) . E Metaphase spreads were prepared as in ( C ) , and the frequency of cells with scattered chromosomes was quantified ( mean ± SD , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

D Metaphase spreads were prepared as in (C), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). E Metaphase spreads were prepared as in (C), and the frequency of cells with scattered chromosomes was quantified (mean ± SD, n = 3). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


F HeLa cells were transfected with control or TIAR si - RNAs , alone or together with Cdc25B siRNA , and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads . The frequency of cells with scattered chromatids was quantified ( mean ± SD , n = 3 independent experiments , approximately 30 metaphase spreads were assessed per experiment and condition ) . G Metaphase spreads were prepared as in ( F ) , and the frequency of cells with chromosomal breaks was quantified ( mean ± SD , n = 3 ) . Data information , statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

F HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA, and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads. The frequency of cells with scattered chromatids was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). G Metaphase spreads were prepared as in (F), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3). Data information , statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0 . 4 µM APH . Cells were fixed at regular time intervals , and p ( S10 ) - H3 - positive cells were quantified by flow cytometry ( mean ± SE , n = 3 ) .

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

A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0.4 µM APH. Cells were fixed at regular time intervals, and p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SE, n = 3).


B HeLa cells transfected with control or TIAR siRNAs were treated with APH for 24 hours . Pan - nuclear γH2AX signals were quantified by HTM ( n = 3 , 2000 cells examined per experiment and condition ) . Each dot represents the signal from one cell , horizontal lines indicate mean values , and the blue area delineates cells above an arbitrarily chosen threshold . Data information : statistical significance was determined by Wilcoxon rank - sum test . ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells transfected with control or TIAR siRNAs were treated with APH for 24 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 2000 cells examined per experiment and condition). Each dot represents the signal from one cell, horizontal lines indicate mean values, and the blue area delineates cells above an arbitrarily chosen threshold. Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


C Chromatin - bound RPA2 signals were quantified by HTM HeLa cells transfected with control or TIAR siRNAs ( n = 3 , 600 cells examined per experiment and condition ) .

O O O O O O O O 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 Chromatin-bound RPA2 signals were quantified by HTM HeLa cells transfected with control or TIAR siRNAs (n = 3, 600 cells examined per experiment and condition).


D HeLa cells were transfected with control or TIAR siRNAs for 72 hours prior to treatment with ATRi ( 5 μM . ) alone or together with Ro3306 ( 5 μM ) for 12 hours . Pan - nuclear γH2AX signals were quantified by HTM ( n = 3 , 1000 cells examined per experiment and condition ) . Data information : statistical significance was determined by Wilcoxon rank - sum test . ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with control or TIAR siRNAs for 72 hours prior to treatment with ATRi (5 μM.) alone or together with Ro3306 (5 μM) for 12 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 1000 cells examined per experiment and condition). Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


E HeLa cells were transfected with control or TIAR siRNAs were treated with ATRi for 48 hours , and the sub - G1 population was quantified by flow cytometry following propidium iodide staining ( mean ± SD , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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

E HeLa cells were transfected with control or TIAR siRNAs were treated with ATRi for 48 hours, and the sub-G1 population was quantified by flow cytometry following propidium iodide staining (mean ± SD, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


F HeLa cells transfected with control or TIAR siRNAs were treated with APH , alone or in combination with Gö6976 ( 1µg / ml ) or UCN - 01 ( 300 nM ) . After 24 hours , p ( S10 ) - H3 - positive cells were quantified by flow cytometry , and expressed relative to the values in the untreated condition ( mean ± SE , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells transfected with control or TIAR siRNAs were treated with APH, alone or in combination with Gö6976 (1µg/ml) or UCN-01 (300 nM). After 24 hours, p(S10)-H3-positive cells were quantified by flow cytometry, and expressed relative to the values in the untreated condition (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


A HeLa cells , untreated or treated for 24 hours with 0 . 4 µM APH , were fixed with methanol and analyzed by IF microscopy after staining with anti - p ( S10 ) - H3 antibody , anti - TIAR antibody and DAPI . Yellow arrows mark focal accumulation of TIAR in GMGs .

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

A HeLa cells, untreated or treated for 24 hours with 0.4 µM APH, were fixed with methanol and analyzed by IF microscopy after staining with anti-p(S10)-H3 antibody, anti-TIAR antibody and DAPI. Yellow arrows mark focal accumulation of TIAR in GMGs.


B IF microscopy was performed as in ( A ) to determine the intensity of the TIAR signal within GMGs ( mean ± SD , n = 3 independent experiments , approximately 100 GMGs in 25 cells were analyzed per experiment and condition ) . C IF microscopy was performed as in ( A ) to determine the area of the TIAR signal within GMGs ( mean ± SD , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

B IF microscopy was performed as in (A) to determine the intensity of the TIAR signal within GMGs (mean ± SD, n = 3 independent experiments, approximately 100 GMGs in 25 cells were analyzed per experiment and condition). C IF microscopy was performed as in (A) to determine the area of the TIAR signal within GMGs (mean ± SD, n = 3). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


D IF microscopy was performed as in ( A ) to quantify the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

D IF microscopy was performed as in (A) to quantify the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


E HeLa cells were processed for IF microscopy as in ( A ) , images depict different cell cycle phases .

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E HeLa cells were processed for IF microscopy as in (A), images depict different cell cycle phases.


HeLa cell were treated for 16 hours with ( F ) the CDK1 inhibitor Ro3306 ( 9 µM ) prior to fixation with methanol and staining with anti - TIAR antibody and DAPI . The graphs show the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

HeLa cell were treated for 16 hours with (F) the CDK1 inhibitor Ro3306 (9 µM) prior to fixation with methanol and staining with anti-TIAR antibody and DAPI. The graphs show the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


HeLa cell were treated for 16 hours with ( G ) ICFR - 193 ( 1 μM ) prior to fixation with methanol and staining with anti - TIAR antibody and DAPI . The graphs show the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) .

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

HeLa cell were treated for 16 hours with (G) ICFR-193 (1 μM) prior to fixation with methanol and staining with anti-TIAR antibody and DAPI. The graphs show the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition).


A - G HeLa cells were treated with 0 . 4 µM APH for 24 hours , fixed and processed for IF microscopy after staining with anti - TIAR antibody , DAPI in combination with ( A ) anti - PCNA - 1 antibody , ( B ) anti - FANCD2 antibody , ( C ) anti - BRCA1 antibody , ( D ) anti - PRP19 antibody , ( E ) anti - Sm ( Y12 ) antibody , ( F ) anti - HuR antibody and ( G ) pS2 - RPOL2 ( H5 ) antibody . Intensity profiles along the white line in the merged image are presented on the right side ; yellow and green arrows mark GMGs .

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A-G HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR antibody, DAPI in combination with (A) anti-PCNA-1 antibody, (B) anti-FANCD2 antibody, (C) anti-BRCA1 antibody, (D) anti-PRP19 antibody, (E) anti-Sm(Y12) antibody, (F) anti-HuR antibody and (G) pS2-RPOL2 (H5) antibody. Intensity profiles along the white line in the merged image are presented on the right side; yellow and green arrows mark GMGs.


A HeLa cells were treated with 0 . 4 µM APH for 24 hours , fixed and processed for IF microscopy after staining with anti - TIAR and anti - CDK1 antibodies . Intensity profiles along the white line in the merged image are presented on the right side ; yellow arrows mark GMGs .

A HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR and anti-CDK1 antibodies. Intensity profiles along the white line in the merged image are presented on the right side; yellow arrows mark GMGs.


B IF microscopy analysis as in ( A ) using anti - Cyclin B antibody .

B IF microscopy analysis as in (A) using anti-Cyclin B antibody.


C HeLa cells transfected with control or TIAR siRNAs were treated with 0 . 4 µM APH 24 hours prior to fixation . Cells were processed for IF microscopy after staining with anti - PRP19 and anti - CDK1 antibodies . Intensity profiles from staining of the same cells with anti - TIAR and anti - CDK1 antibodies are depicted on the right side .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells transfected with control or TIAR siRNAs were treated with 0.4 µM APH 24 hours prior to fixation. Cells were processed for IF microscopy after staining with anti-PRP19 and anti-CDK1 antibodies. Intensity profiles from staining of the same cells with anti-TIAR and anti-CDK1 antibodies are depicted on the right side.


D CDK1 was immunoprecipitated from HeLa cells transfected with control or TIAR siRNAs , tested for in vitro kinase activity using recombinant histone H1 as substrate , and visualized by Western blot analysis and autoradiography . The mean CDK1 activity ± SE was quantified from n = 5 experiments . Data information statistical significance was determined by unpaired Student ' s t - test . ; * , p < 0 . 05 ; * * * , p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 CDK1 was immunoprecipitated from HeLa cells transfected with control or TIAR siRNAs, tested for in vitro kinase activity using recombinant histone H1 as substrate, and visualized by Western blot analysis and autoradiography. The mean CDK1 activity ± SE was quantified from n = 5 experiments. Data information statistical significance was determined by unpaired Student's t-test. ; *, p < 0.05; ***, p < 0.001.


E CDK2 activity was analyzed following immunoprecipitation as in ( D ) ( mean ± SE , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test . * , p < 0 . 05 ; * * * , p < 0 . 001 .

E CDK2 activity was analyzed following immunoprecipitation as in (D) (mean ± SE, n = 3). Data information statistical significance was determined by unpaired Student's t-test. *, p < 0.05; ***, p < 0.001.


F HeLa cells were transfected with control or TIAR siRNAs for 72 hours before measuring expression levels of p ( S22 ) - Lamin A by Western blot analysis ( mean ± SE , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test . ; * , p < 0 . 05 ; * * * , p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with control or TIAR siRNAs for 72 hours before measuring expression levels of p(S22)-Lamin A by Western blot analysis (mean ± SE, n = 3). Data information statistical significance was determined by unpaired Student's t-test. ; *, p < 0.05; ***, p < 0.001.


G p ( S22 ) - Lamin A levels were analyzed by HTM in si - RNA transfected HeLa cells ( n = 3 , 1000 cells examined per experiment and condition ) . Data information statistical significance was determined by Wilcoxon rank - sum test ; * , p < 0 . 05 ; * * * , p < 0 . 001 .

G p(S22)-Lamin A levels were analyzed by HTM in si-RNA transfected HeLa cells (n = 3, 1000 cells examined per experiment and condition). Data information statistical significance was determined by Wilcoxon rank-sum test; *, p < 0.05; ***, p < 0.001.


HeLa cells were co - transfected with Myc empty vector ( Myc ) , Myc - tagged FUNDC1 ( F1 - Myc ) or Myc - tagged FUNDC1 - ΔLIR mutant ( ΔLIR - Myc ) and Mito - OM - cherry , which localizes to the mitochondrial outer membrane , and immunostained with fluorescent anti - p62 and anti - Myc antibodies . Scale bar = 10 μm .

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

HeLa cells were co-transfected with Myc empty vector (Myc), Myc-tagged FUNDC1 (F1-Myc) or Myc-tagged FUNDC1-ΔLIR mutant (ΔLIR-Myc) and Mito-OM-cherry, which localizes to the mitochondrial outer membrane, and immunostained with fluorescent anti-p62 and anti-Myc antibodies. Scale bar=10 μm.


HeLa cells were co - transfected with Myc , F1 - Myc or ΔLIR - Myc and Mito - OM - cherry and immunostained with anti - Ubiquitin ( Ub ) and anti - Myc antibodies . Scale bar = 10 µm .

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

HeLa cells were co-transfected with Myc, F1-Myc or ΔLIR-Myc and Mito-OM-cherry and immunostained with anti-Ubiquitin (Ub) and anti-Myc antibodies. Scale bar=10 µm.


Fractionation analysis of HeLa cells transfected with Myc , F1 - Myc or ΔLIR - Myc . The loading controls ( L . C . ) were α - Tubulin for total cell lysate ( TCL ) and cytosolic fraction ( Cyto ) , and HSP60 for mitochondrial fraction ( Mito ) .

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

Fractionation analysis of HeLa cells transfected with Myc, F1-Myc or ΔLIR-Myc. The loading controls (L.C.) were α-Tubulin for total cell lysate (TCL) and cytosolic fraction (Cyto), and HSP60 for mitochondrial fraction (Mito).


HeLa cells transfected with Myc or Myc / His6 double - tagged F1 - Myc - His were lysed and precipitated with Ni - NTA beads . Samples were then subjected to SDS - PAGE . The differential band ( upper arrow ) was isolated and subjected to mass spectral analysis . The identified proteins with a score higher than 50 are listed .

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

HeLa cells transfected with Myc or Myc/His6 double-tagged F1-Myc-His were lysed and precipitated with Ni-NTA beads. Samples were then subjected to SDS-PAGE. The differential band (upper arrow) was isolated and subjected to mass spectral analysis. The identified proteins with a score higher than 50 are listed.


Co - IP of endogenous HSC70 with F1 - Myc or ΔLIR - Myc . α - Myc , anti - Myc

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

Co-IP of endogenous HSC70 with F1-Myc or ΔLIR-Myc. α-Myc, anti-Myc antibody


Co - IP of HSC70 with FUNDC1 . α - F1 , anti - FUNDC1 antibody . rIgG , control rabbit IgG .

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

Co-IP of HSC70 with FUNDC1. α-F1, anti-FUNDC1 antibody. rIgG, control rabbit IgG.


Co - IP analysis of endogenous FUNDC1 with Flag - HSP70 or Flag - HSC70 . α - Flag , anti - Flag antibody . Flag ， empty vector .

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

Co-IP analysis of endogenous FUNDC1 with Flag-HSP70 or Flag-HSC70. α-Flag, anti-Flag antibody. Flag，empty vector.


HeLa cells were co - transfected with Myc , F1 - Myc or ΔLIR - Myc and Mito - OM - cherry and immunostained with anti - HSC70 and anti - Myc antibodies . Scale bar = 10 µm .

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

HeLa cells were co-transfected with Myc, F1-Myc or ΔLIR-Myc and Mito-OM-cherry and immunostained with anti-HSC70 and anti-Myc antibodies. Scale bar=10 µm.


Fractionation analysis of HeLa cells transfected with Myc or F1 - Myc .

Fractionation analysis of HeLa cells transfected with Myc or F1-Myc.


HeLa cells were co - transfected with Ub - R - GFP and Myc or F1 - Myc and immunostained with fluorescent anti - Myc and anti - GFP ( α - GFP ) antibodies . The α - GFP signals in F1 - Myc transfected cells were lowered to avoid the overexposure of the mitochondrially localized R - GFP , making such signals of the equally distributed R - GFP invisible . Green arrowheads show the mitochondrially localized R - GFP proteins , which were more abundant than the equally distributed R - GFP and showed stronger R - GFP fluorescence , white arrowheads show the mitochondrially localized R - GFP proteins without stronger R - GFP fluorescence , although abundant . Scale bar = 10 µm .

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

HeLa cells were co-transfected with Ub-R-GFP and Myc or F1-Myc and immunostained with fluorescent anti-Myc and anti-GFP (α-GFP) antibodies. The α-GFP signals in F1-Myc transfected cells were lowered to avoid the overexposure of the mitochondrially localized R-GFP, making such signals of the equally distributed R-GFP invisible. Green arrowheads show the mitochondrially localized R-GFP proteins, which were more abundant than the equally distributed R-GFP and showed stronger R-GFP fluorescence, white arrowheads show the mitochondrially localized R-GFP proteins without stronger R-GFP fluorescence, although abundant. Scale bar=10 µm.


Fractionation analysis of HeLa cells co - transfected with Ub - R - GFP and Myc or F1 - Myc .

Fractionation analysis of HeLa cells co-transfected with Ub-R-GFP and Myc or F1-Myc.


HeLa cells were co - transfected with Ub - R - GFP and F1 - Myc . Mitochondria were isolated and then treated with or without proteinase K ( Pro . K ) , or proteinase K in combination with digitonin or Triton X - 100 ( TX - 100 ) . Samples were then subjected to Immunoblot analysis . HSP60 , TIM23 and TOM20 are representatives of mitochondrial matrix - , mitochondrial inner membrane - and mitochondrial outer membrane - localized proteins , respectively .

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

HeLa cells were co-transfected with Ub-R-GFP and F1-Myc. Mitochondria were isolated and then treated with or without proteinase K (Pro.K), or proteinase K in combination with digitonin or Triton X-100 (TX-100). Samples were then subjected to Immunoblot analysis. HSP60, TIM23 and TOM20 are representatives of mitochondrial matrix-, mitochondrial inner membrane- and mitochondrial outer membrane-localized proteins, respectively.


Fractionation analysis of HeLa cells stably transfected with control ( Ctrl ) shRNA vector or HSC70 shRNA and transiently co - transfected with Ub - R - GFP , Flag or Flag - HSC70 ( shRNA - resistant ) and Myc , or F1 - Myc , as 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 O O O O O O O O O O

Fractionation analysis of HeLa cells stably transfected with control (Ctrl) shRNA vector or HSC70 shRNA and transiently co-transfected with Ub-R-GFP, Flag or Flag-HSC70 (shRNA-resistant) and Myc, or F1-Myc, as indicated.


Fractionation analysis of the HeLa cells transfected with Ub - R - GFP and treated with DMSO ( control vehicle ) or 10 μM MG132 for 4 hours .

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

Fractionation analysis of the HeLa cells transfected with Ub-R-GFP and treated with DMSO (control vehicle) or 10 μM MG132 for 4 hours.


Proteinase K protection analysis of the mitochondria isolated from the HeLa cells transfected with Ub - R - GFP and treated with MG132 .

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

Proteinase K protection analysis of the mitochondria isolated from the HeLa cells transfected with Ub-R-GFP and treated with MG132.


Proteinase K protection analysis of the mitochondria isolated from HeLa cells co - transfected with Ub - R - GFP and Ctrl , TOM20 , TOM22 or TOM70 shRNA and treated with MG132 .

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

Proteinase K protection analysis of the mitochondria isolated from HeLa cells co-transfected with Ub-R-GFP and Ctrl, TOM20, TOM22 or TOM70 shRNA and treated with MG132.


Fractionation analysis of HeLa cells stably transfected with Ctrl or LONP1 shRNA and transiently co - transfected with Ub - R - GFP and Myc or LONP1 ( shRNA - resistant ) and then treated with DMSO or MG132 .

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

Fractionation analysis of HeLa cells stably transfected with Ctrl or LONP1 shRNA and transiently co-transfected with Ub-R-GFP and Myc or LONP1 (shRNA-resistant) and then treated with DMSO or MG132.


Fractionation analysis of HeLa cells stably transfected with Ctrl shRNA , F1 shRNA , or F1 shRNA together with shRNA - resistant F1 - Myc or ΔLIR - Myc , and transiently transfected with Ub - R - GFP and then treated with DMSO or MG132 .

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

Fractionation analysis of HeLa cells stably transfected with Ctrl shRNA, F1 shRNA, or F1 shRNA together with shRNA-resistant F1-Myc or ΔLIR-Myc, and transiently transfected with Ub-R-GFP and then treated with DMSO or MG132.


Fractionation analysis of HeLa cells stably transfected with Ctrl or HSC70 shRNA and transiently co - transfected with Ub - R - GFP and Flag or shRNA - resistant Flag - HSC70 and then treated with DMSO or MG132 .

Fractionation analysis of HeLa cells stably transfected with Ctrl or HSC70 shRNA and transiently co-transfected with Ub-R-GFP and Flag or shRNA-resistant Flag-HSC70 and then treated with DMSO or MG132.


Fractionation analysis of HeLa cells stably transfected with Ctrl or HSP70 shRNA and transiently co - transfected with Ub - R - GFP and Flag or shRNA - resistant Flag - HSP70 and then treated with DMSO or MG132 .

Fractionation analysis of HeLa cells stably transfected with Ctrl or HSP70 shRNA and transiently co-transfected with Ub-R-GFP and Flag or shRNA-resistant Flag-HSP70 and then treated with DMSO or MG132.


Mitochondria were isolated from the untransfected cells , cytosols were isolated from the cells transfected with GFP or Ub - R - GFP and treated with DMSO or MG132 for 8 hours , the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis .

O O O O 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

Mitochondria were isolated from the untransfected cells, cytosols were isolated from the cells transfected with GFP or Ub-R-GFP and treated with DMSO or MG132 for 8 hours, the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis.


Mitochondria isolated from the untransfected cells were resuspended in the cytosol isolated from the cells transfected with Ub - R - GFP and incubated at RT for I hour , the mitochondria were then isolated from the in vitro system , then treated with or without proteinase K and subjected to immunoblot analysis .

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

Mitochondria isolated from the untransfected cells were resuspended in the cytosol isolated from the cells transfected with Ub-R-GFP and incubated at RT for I hour, the mitochondria were then isolated from the in vitro system, then treated with or without proteinase K and subjected to immunoblot analysis.


Mitochondria were isolated from the cells transfected with ctrl shRNA , F1 shRNA or F1 shRNA together with F1 - Myc , cytosols were isolated from the cells transfected with Ub - R - GFP , the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis .

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

Mitochondria were isolated from the cells transfected with ctrl shRNA, F1 shRNA or F1 shRNA together with F1-Myc, cytosols were isolated from the cells transfected with Ub-R-GFP, the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis.


Cytosol isolated from the cells transfected with Ub - R - GFP were mixed with the cytosols isolated from the cells transfected with ctrl shRNA , HSC70 shRNA or HSC70 shRNA together with Flag - HSC70 , mitochondria were isolated from the untransfected cells , the isolated mitochondria and cytosol mixtures were then subjected to in vitro protein import analysis .

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

Cytosol isolated from the cells transfected with Ub-R-GFP were mixed with the cytosols isolated from the cells transfected with ctrl shRNA, HSC70 shRNA or HSC70 shRNA together with Flag-HSC70, mitochondria were isolated from the untransfected cells, the isolated mitochondria and cytosol mixtures were then subjected to in vitro protein import analysis.


Co - IP of HSC70 with FUNDC1 in HeLa cells treated with MG132 as indicated .

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

Co-IP of HSC70 with FUNDC1 in HeLa cells treated with MG132 as indicated.


Immunoblot analysis of the HeLa cells transfected with or without HA - Bcl - xL and treated with DMSO or MG132 for 6 hours .

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

Immunoblot analysis of the HeLa cells transfected with or without HA-Bcl-xL and treated with DMSO or MG132 for 6 hours.


Ni2 + - NTA precipitation of HeLa cells transfected with indicated vectors . S13A - Myc - His , Myc / His6 double - tagged FUNDC1 - S13A mutant .

Ni2+-NTA precipitation of HeLa cells transfected with indicated vectors. S13A-Myc-His, Myc/His6 double-tagged FUNDC1-S13A mutant.


D , E . Fractionation analysis of HeLa cells transfected with indicated vectors and treated with DMSO or MG132 . Quantification is shown in ( E ) .

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

D, E. Fractionation analysis of HeLa cells transfected with indicated vectors and treated with DMSO or MG132. Quantification is shown in (E).


HeLa cells were transfected with GFP - p62 , treated with DMSO or 10 μM MG132 for 8 hours and then immunostained with anti - Ub and anti - γ - Tubulin antibodies . Note that the aggresome ( blue arrowhead ) , rather than the MAPAs ( white arrowheads ) , colocalizes with γ - Tubulin . Scale bar = 10 µm .

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

HeLa cells were transfected with GFP-p62, treated with DMSO or 10 μM MG132 for 8 hours and then immunostained with anti-Ub and anti-γ-Tubulin antibodies. Note that the aggresome (blue arrowhead), rather than the MAPAs (white arrowheads), colocalizes with γ-Tubulin. Scale bar=10 µm.


B , C . HeLa cells were stably transfected with the indicated vectors , treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm .

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

B, C. HeLa cells were stably transfected with the indicated vectors, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm.


B , C . HeLa cells were stably transfected with the indicated vectors , treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm . Quantification is show in ( C ) .

O O O O O O O O O O O O O O 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. HeLa cells were stably transfected with the indicated vectors, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (C).


D , E . HeLa cells were transfected with Ctrl , TOM20 , TOM22 or TOM70 shRNA vectors including nuclear localized GFP ( N - GFP ) expressing elements , then treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm . Quantification is show in ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E. HeLa cells were transfected with Ctrl, TOM20, TOM22 or TOM70 shRNA vectors including nuclear localized GFP (N-GFP) expressing elements, then treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (E).


F , G . HeLa cells were transfected with GFP or GFP - Bcl - xL , then treated with MG132 and immunostained with anti - p62 antibodies . Scale bar = 10 µm . Quantification is show in ( G ) .

O O O O O O O O O O O O O O O O O O O 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, G. HeLa cells were transfected with GFP or GFP-Bcl-xL, then treated with MG132 and immunostained with anti-p62 antibodies. Scale bar=10 µm. Quantification is show in (G).


H , I . HeLa cells were stably transfected with Ctrl or LONP1 shRNA , treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm . Quantification is show in ( I ) .

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

H, I. HeLa cells were stably transfected with Ctrl or LONP1 shRNA, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (I).


csCLEM analysis of cells transfected with GFP - p62 and treated with MG132 . Red arrowheads show the dark red fluorescent beads used to correlate the images from light microscopy ( LM ) and transmission electron microscopy ( TEM ) . The blue arrowhead shows a MAPA observed under LM and TEM .

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

csCLEM analysis of cells transfected with GFP-p62 and treated with MG132. Red arrowheads show the dark red fluorescent beads used to correlate the images from light microscopy (LM) and transmission electron microscopy (TEM). The blue arrowhead shows a MAPA observed under LM and TEM.


TEM analysis of HeLa cells treated with DMSO or MG132 . Note the layered structures ( red arrowheads ) in the mitochondrial matrix ( ii , iii ) , budding from mitochondria ( iv ) , attached to mitochondria ( v - vi ) , away from mitochondria ( vii ) or inside the MAPAs ( viii , ix ) in MG132 - treated cells . Scale bar = 500 nm . M , mitochondrion . N , nucleus .

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

TEM analysis of HeLa cells treated with DMSO or MG132. Note the layered structures (red arrowheads) in the mitochondrial matrix (ii, iii), budding from mitochondria (iv), attached to mitochondria (v-vi), away from mitochondria (vii) or inside the MAPAs (viii, ix) in MG132-treated cells. Scale bar=500 nm. M, mitochondrion. N, nucleus.


Immunoblot analysis of the TX - 100 - soluble and - insoluble fractions of HeLa cells treated with MG132 as indicated .

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

Immunoblot analysis of the TX-100-soluble and -insoluble fractions of HeLa cells treated with MG132 as indicated.


Immunoblot analysis of the TX - 100 - soluble and - insoluble fractions of HeLa cells transfected with Ub - R - GFP and treated with MG132 as 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 B-CONTROLLED_VAR O O O

Immunoblot analysis of the TX-100-soluble and -insoluble fractions of HeLa cells transfected with Ub-R-GFP and treated with MG132 as indicated.


HeLa cells were co - transfected with Mito - OM - cherry and Mito - MTX - GFP , then treated with DMSO or MG132 and immunostained with anti - p62 antibody . Note the colocalization of a p62 punctum with a structure that is positive for Mito - OM - cherry but not for Mito - MTX - GFP ( white arrowheads ) in MG132 - treated cells . Scale bar = 10 µm .

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

HeLa cells were co-transfected with Mito-OM-cherry and Mito-MTX-GFP, then treated with DMSO or MG132 and immunostained with anti-p62 antibody. Note the colocalization of a p62 punctum with a structure that is positive for Mito-OM-cherry but not for Mito-MTX-GFP (white arrowheads) in MG132-treated cells. Scale bar=10 µm.


HeLa cells were transfected with Mito - MTX - GFP , then treated with DMSO or MG132 and immunostained with anti - p62 and anti - TIM23 antibodies . Note the colocalization of a p62 punctum with a structure that is positive for TIM3 but not for Mito - MTX - GFP ( white arrowhead ) in MG132 - treated cells . Scale bar = 10 µm .

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

HeLa cells were transfected with Mito-MTX-GFP, then treated with DMSO or MG132 and immunostained with anti-p62 and anti-TIM23 antibodies. Note the colocalization of a p62 punctum with a structure that is positive for TIM3 but not for Mito-MTX-GFP (white arrowhead) in MG132-treated cells. Scale bar=10 µm.


HeLa cells were co - transfected with Mito - MTX - GFP and OTC - Myc or OTC - Δ - Myc , treated with DMSO or MG132 and immunostained with anti - Myc and anti - p62 antibodies . Note that the p62 - labled MAPAs were colocalized with OTC - ∆ ( red arrowheads ) , but not with OTC ( white arrowheads ) in the MG132 treated cells . Scale bar = 10 µm .

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

HeLa cells were co-transfected with Mito-MTX-GFP and OTC-Myc or OTC-Δ-Myc, treated with DMSO or MG132 and immunostained with anti-Myc and anti-p62 antibodies. Note that the p62-labled MAPAs were colocalized with OTC-∆ (red arrowheads), but not with OTC (white arrowheads) in the MG132 treated cells. Scale bar=10 µm.


TEM analysis of HeLa cells transfected with indicated vectors , treated with MG132 and stained with DAB . Blue arrowheads show MAPAs , red arrowheads show electron - dense Apex signals . Scale bar = 500 nm . M , mitochondrion .

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

TEM analysis of HeLa cells transfected with indicated vectors, treated with MG132 and stained with DAB. Blue arrowheads show MAPAs, red arrowheads show electron-dense Apex signals. Scale bar=500 nm. M, mitochondrion.


Time - lapse analysis of live HeLa cells transfected with OTC - Δ - GFP and Mito - MTX - DsRed and treated with MG132 . White arrowheads show a OTC - Δ - GFP - positive Mito - MTX - DsRed - negative structure that eventually segregated from the mitochondrial network .

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

Time-lapse analysis of live HeLa cells transfected with OTC-Δ-GFP and Mito-MTX-DsRed and treated with MG132. White arrowheads show a OTC-Δ-GFP-positive Mito-MTX-DsRed-negative structure that eventually segregated from the mitochondrial network.


Immunoblot analysis of the MG132 - induced deposition of FUNDC1 , TIM23 and p62 in HeLa cells transfected with Ctrl or DLP1 shRNA .

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

Immunoblot analysis of the MG132-induced deposition of FUNDC1, TIM23 and p62 in HeLa cells transfected with Ctrl or DLP1 shRNA.


C , D . Immunoblot analysis of the MG132 - induced deposition of FUNDC1 in cells transfected with ctrl shRNA , FIS1 shRNA or FIS1 shRNA together with shRNA - resistant Flag - FIS1 . Quantification is shown in ( D ) .

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C, D. Immunoblot analysis of the MG132-induced deposition of FUNDC1 in cells transfected with ctrl shRNA, FIS1 shRNA or FIS1 shRNA together with shRNA-resistant Flag-FIS1. Quantification is shown in (D).


E , F . HeLa cells were stably transfected with Ctrl or FIS1 shRNA , treated with MG132 and immunostained with indicated antibodies . Scale bar = 10 µm . Quantification is shown in ( F ) .

E, F. HeLa cells were stably transfected with Ctrl or FIS1 shRNA, treated with MG132 and immunostained with indicated antibodies. Scale bar=10 µm. Quantification is shown in (F).


G , H . Fractionation analysis of HeLa cells stably transfected with Ctrl or FIS1 shRNA , then transiently co - transfected with Ub - R - GFP and Flag or Flag - FIS1 and treated with DMSO or MG132 . Quantification is shown in ( H ) .

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G, H. Fractionation analysis of HeLa cells stably transfected with Ctrl or FIS1 shRNA, then transiently co-transfected with Ub-R-GFP and Flag or Flag-FIS1 and treated with DMSO or MG132. Quantification is shown in (H).


HeLa cells were transfected with GFP - LC3 , treated with DMSO or 10 μM MG132 for 8 hours and immunostained with anti - p62 antibody . Scale bar = 10 µm .

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HeLa cells were transfected with GFP-LC3, treated with DMSO or 10 μM MG132 for 8 hours and immunostained with anti-p62 antibody. Scale bar=10 µm.


Immunoblot analysis of the TCL of HeLa cells treated with MG132 as indicated .

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Immunoblot analysis of the TCL of HeLa cells treated with MG132 as indicated.


Immunoblot analysis of the TCL of HeLa cells stably transfected with Ctrl or ATG5 shRNA and treated with MG132 as indicated .

Immunoblot analysis of the TCL of HeLa cells stably transfected with Ctrl or ATG5 shRNA and treated with MG132 as indicated.


Immunoblot analysis of the TCL of HeLa cells stably transfected with the indicated vectors and treated with MG132 as indicated . Quantification of the results from panels ( D ) and ( E ) . 3 replicates were analyzed using Student ' s 2 - tailed t - test and are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Immunoblot analysis of the TCL of HeLa cells stably transfected with the indicated vectors and treated with MG132 as indicated.   Quantification of the results from panels (D) and (E). 3 replicates were analyzed using Student's 2-tailed t-test and are presented as the means ± SEM. * p<0.05, ** p<0.01, *** p<0.001.


HeLa cells were stably transfected with the indicated vectors , transiently transfected with GFP - LC3 , then treated with 10 μM MG132 for 8 hours and immunostained with anti - p62 antibody . Scale bar = 10 µm .

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HeLa cells were stably transfected with the indicated vectors, transiently transfected with GFP-LC3, then treated with 10 μM MG132 for 8 hours and immunostained with anti-p62 antibody. Scale bar=10 µm.


TEM analysis of F1 shRNA / F1 - Myc or F1 shRNA / ΔLIR - Myc cells treated with MG132 for 8 hours . Yellow arrowheads show the MAPAs . Blue arrows show the places where membrane is absent . White arrowheads show the layered aggregates budding from mitochondria . Scale bar = 500 nm ; M , mitochondrion .

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TEM analysis of F1 shRNA/F1-Myc or F1 shRNA/ΔLIR-Myc cells treated with MG132 for 8 hours. Yellow arrowheads show the MAPAs. Blue arrows show the places where membrane is absent. White arrowheads show the layered aggregates budding from mitochondria. Scale bar=500 nm; M, mitochondrion.


A , B . HeLa cells were treated with DMSO or 10 μM MG132 for 8 hours , then washed with fresh medium three times and cultured for another 3 days . Cells were then fixed and subjected to SA - β - gal staining . The senescent cells show SA - β - gal - positive staining ( Blue ) . Quantification is shown in ( B ) .

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A, B. HeLa cells were treated with DMSO or 10 μM MG132 for 8 hours, then washed with fresh medium three times and cultured for another 3 days. Cells were then fixed and subjected to SA-β-gal staining. The senescent cells show SA-β-gal-positive staining (Blue). Quantification is shown in (B).


C , D . Immunoblot analysis of HeLa cells treated with DMSO or MG132 for 8 hours , then washed and cultured for the indicated times . Quantification is shown in ( D ) .

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C, D. Immunoblot analysis of HeLa cells treated with DMSO or MG132 for 8 hours, then washed and cultured for the indicated times. Quantification is shown in (D).


E , Quantification of SA - β - gal staining in HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C ( C . C ) for another 3 days .

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E, Quantification of SA-β-gal staining in HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C (C.C) for another 3 days.


F . HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C ( C . C ) for another 3 days . Immunoblot analysis of the cells is shown in ( F ) .

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F. HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C (C.C) for another 3 days. Immunoblot analysis of the cells is shown in (F).


Quantification of SA - β - gal staining of HeLa cells stably transfected with the indicated vectors , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1 - Myc or ΔLIR - Myc , although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells .

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Quantification of SA-β-gal staining of HeLa cells stably transfected with the indicated vectors, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1-Myc or ΔLIR-Myc, although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells.


H . HeLa cells stably transfected with the indicated vectors , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( H ) . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1 - Myc or ΔLIR - Myc , although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells .

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H. HeLa cells stably transfected with the indicated vectors, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (H). The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1-Myc or ΔLIR-Myc, although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells.


Quantification of MG132 - induced SA - β - gal staining in HeLa cells stably transfected with Ctrl , HSP70 or HSC70 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown , although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells . Both p53 and p21 were up - regulated in HSP70 knockdown cells treated with or without MG132 .

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Quantification of MG132-induced SA-β-gal staining in HeLa cells stably transfected with Ctrl, HSP70 or HSC70 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown, although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells. Both p53 and p21 were up-regulated in HSP70 knockdown cells treated with or without MG132.


J . HeLa cells stably transfected with Ctrl , HSP70 or HSC70 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( J ) . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown , although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells . Both p53 and p21 were up - regulated in HSP70 knockdown cells treated with or without MG132 .

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J. HeLa cells stably transfected with Ctrl, HSP70 or HSC70 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (J). The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown, although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells. Both p53 and p21 were up-regulated in HSP70 knockdown cells treated with or without MG132.


HeLa cells stably transfected with tetracycline ( Tet ) - inducible Bcl - xL expression vector were incubated in DMEM containing Tet or not for 12 hours , then treated with DMSO or 10 μM MG132 for 8 hours , then washed and incubated in fresh medium for another 3 days , Cells were then subjected to SA - β - gal staining . The induced Bcl - xL was detectable even 3 days after treatment withdrawal , because this protein is stable .

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HeLa cells stably transfected with tetracycline (Tet)-inducible Bcl-xL expression vector were incubated in DMEM containing Tet or not for 12 hours, then treated with DMSO or 10 μM MG132 for 8 hours, then washed and incubated in fresh medium for another 3 days, Cells were then subjected to SA-β-gal staining. The induced Bcl-xL was detectable even 3 days after treatment withdrawal, because this protein is stable.


L . HeLa cells stably transfected with tetracycline ( Tet ) - inducible Bcl - xL expression vector were incubated in DMEM containing Tet or not for 12 hours , then treated with DMSO or 10 μM MG132 for 8 hours , then washed and incubated in fresh medium for another 3 days , Immunoblot analysis of the cells is shown in ( L ) . The induced Bcl - xL was detectable even 3 days after treatment withdrawal , because this protein is stable .

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L. HeLa cells stably transfected with tetracycline (Tet)-inducible Bcl-xL expression vector were incubated in DMEM containing Tet or not for 12 hours, then treated with DMSO or 10 μM MG132 for 8 hours, then washed and incubated in fresh medium for another 3 days, Immunoblot analysis of the cells is shown in (L). The induced Bcl-xL was detectable even 3 days after treatment withdrawal, because this protein is stable.


Quantification of the SA - β - gal staining of HeLa cells stably transfected with Ctrl or LONP1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days .

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Quantification of the SA-β-gal staining of HeLa cells stably transfected with Ctrl or LONP1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days.


, N . HeLa cells stably transfected with Ctrl or LONP1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( N ) .

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, N. HeLa cells stably transfected with Ctrl or LONP1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (N).


Quantification of the SA - β - gal staining of HeLa cells stably transfected with Ctrl or FIS1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days .

Quantification of the SA-β-gal staining of HeLa cells stably transfected with Ctrl or FIS1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days.


P . HeLa cells stably transfected with Ctrl or FIS1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( P ) .

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P. HeLa cells stably transfected with Ctrl or FIS1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (P).


Single - cell expression data in pseudotime of central regulators of naïve pluripotency , epiblast marker genes , glycolytic regulators , and genes with key functions in oxidative metabolism .

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Single-cell expression data in pseudotime of central regulators of naïve pluripotency, epiblast marker genes, glycolytic regulators, and genes with key functions in oxidative metabolism.


Flow cytometry analysis of Rex1 - GFPd2 cells following addition of 10mM 2 - DG during the 48h EpiLC induction . Representative GFP intensity distributions are depicted . Average proportions of Rex1 - GFPd2 positive ( GFP + ) cells are quantified from two independent biological replicates .

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Flow cytometry analysis of Rex1-GFPd2 cells following addition of 10mM 2-DG during the 48h EpiLC induction. Representative GFP intensity distributions are depicted. Average proportions of Rex1-GFPd2 positive (GFP+) cells are quantified from two independent biological replicates.


Expression analysis by qRT - PCR of naïve pluripotency and epiblast marker genes in bulk 48h cells after 10mM 2 - DG treatment . Relative expression levels , normalized to control culture conditions , are shown . Graphs represent averages from triplicate ( duplicate for Klf4 and Tfcp2l1 ) independent biological experiments .

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Expression analysis by qRT-PCR of naïve pluripotency and epiblast marker genes in bulk 48h cells after 10mM 2-DG treatment. Relative expression levels, normalized to control culture conditions, are shown. Graphs represent averages from triplicate (duplicate for Klf4 and Tfcp2l1) independent biological experiments.


Colony - forming ability following 10mM 2 - DG supplementation during EpiLC stimulation . Representative images of alkaline phosphatase ( AP ) - stained colonies are displayed . Scale bar , 250 um . The average colony formation , normalized to control culture conditions , quantified from two independent biological replicates , is shown .

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Colony-forming ability following 10mM 2-DG supplementation during EpiLC stimulation. Representative images of alkaline phosphatase (AP)-stained colonies are displayed. Scale bar, 250 um. The average colony formation, normalized to control culture conditions, quantified from two independent biological replicates, is shown.


Pseudotime expression profiles for the αKG - regulating enzymes Idh2 and Dlst during the transition from naïve to primed pluripotency . Tricarboxylic acid ( TCA ) cycle enzymes and metabolites produced within the TCA cycle are illustrated .

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Pseudotime expression profiles for the αKG-regulating enzymes Idh2 and Dlst during the transition from naïve to primed pluripotency. Tricarboxylic acid (TCA) cycle enzymes and metabolites produced within the TCA cycle are illustrated.


Representative flow cytometry profiles of Rex1 - GFPd2 cells following 4mM dm - αKG supplementation during the EpiLC induction are depicted . Graphs show average fractions of Rex1 - GFPd2 positive ( GFP + ) cells from six independent biological assays .

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Representative flow cytometry profiles of Rex1-GFPd2 cells following 4mM dm-αKG supplementation during the EpiLC induction are depicted. Graphs show average fractions of Rex1-GFPd2 positive (GFP+) cells from six independent biological assays.


qRT - PCR analysis of naïve pluripotency regulators and epiblast marker genes following EpiLC stimulation in the presence of 4mM dm - αKG . Expression data are normalized to control culture conditions and represent averages from five biological replicates in bulk 48h cells .

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qRT-PCR analysis of naïve pluripotency regulators and epiblast marker genes following EpiLC stimulation in the presence of 4mM dm-αKG. Expression data are normalized to control culture conditions and represent averages from five biological replicates in bulk 48h cells.


Colony forming ability succeeding 4mM dm - αKG treatment during the 48h EpiLC induction . Characteristic images of AP - stained colonies are shown . Scale bar , 250 um . Colony formation is normalized to control - treated cells and quantified from quadruplicate experiments .

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Colony forming ability succeeding 4mM dm-αKG treatment during the 48h EpiLC induction. Characteristic images of AP-stained colonies are shown. Scale bar, 250 um. Colony formation is normalized to control-treated cells and quantified from quadruplicate experiments.


Representative super - resolution images of TOM - 20 immune - labelled mitochondria in ESCs following 48h culture in 2i / Lif / KSR media , and EpiLC - inducing conditions in the presence of 4mM dm - αKG and DMSO , respectively , are displayed . Scale bar , 3um .

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Representative super-resolution images of TOM-20 immune-labelled mitochondria in ESCs following 48h culture in 2i/Lif/KSR media, and EpiLC-inducing conditions in the presence of 4mM dm-αKG and DMSO, respectively, are displayed. Scale bar, 3um.


10 - day culture of Rex1 - GFPd2 cells in N2B27 / Lif / KSR with 4mM dm - αKG and DMSO , respectively , with passaging every 2 . 5 - days . ( F ) Characteristic bright field images of Rex1 - GFPd2 cells after 10 days of culture in 2i / Lif / KSR and N2B27 / Lif / KSR , in the presence of dm - αKG and DMSO , respectively . Scale bar , 10um .

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10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. (F) Characteristic bright field images of Rex1-GFPd2 cells after 10 days of culture in 2i/Lif/KSR and N2B27/Lif/KSR, in the presence of dm-αKG and DMSO, respectively. Scale bar, 10um.


10 - day culture of Rex1 - GFPd2 cells in N2B27 / Lif / KSR with 4mM dm - αKG and DMSO , respectively , with passaging every 2 . 5 - days . Flow cytometer - based quantification of Rex1 - GFPd2 positive ( GFP + ) cells . Representative GFP intensity distributions are displayed . The average fractions of GFP + cells are measured from duplicate experiments .

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10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. Flow cytometer-based quantification of Rex1-GFPd2 positive (GFP+) cells. Representative GFP intensity distributions are displayed. The average fractions of GFP+ cells are measured from duplicate experiments.


10 - day culture of Rex1 - GFPd2 cells in N2B27 / Lif / KSR with 4mM dm - αKG and DMSO , respectively , with passaging every 2 . 5 - days . qRT - PCR analysis of naïve pluripotency and differentiation markers in bulk cells harvested at 2 . 5 day - intervals during the 10 - day culture in N2B27 / Lif / KSR with dm - αKG or DMSO . Expression data are normalized to time - matched ESCs in 2i / Lif / KSR - culture conditions and are averaged over two independent biological experiments .

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10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. qRT-PCR analysis of naïve pluripotency and differentiation markers in bulk cells harvested at 2.5 day-intervals during the 10-day culture in N2B27/Lif/KSR with dm-αKG or DMSO. Expression data are normalized to time-matched ESCs in 2i/Lif/KSR-culture conditions and are averaged over two independent biological experiments.


FACS analysis of Prdm1 - GFP positive ( GFP + ) cells in day - 4 embryoids specified in the presence of 4mM dm - αKG and PGC cytokines . Representative flow cytometer profiles are depicted . Graphs show the average fractions of GFP + cells from duplicate experiments . P1 - GFP , Prdm1 - GFP .

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FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 embryoids specified in the presence of 4mM dm-αKG and PGC cytokines. Representative flow cytometer profiles are depicted. Graphs show the average fractions of GFP+ cells from duplicate experiments. P1-GFP, Prdm1-GFP.


FACS analysis of Prdm1 - GFP positive ( GFP + ) cells in day - 2 embryoids aggregated under addition of LIF ( 10ng ml - 1 ) and BMP4 ( 500ng ml - 1 ) , dm - αKG ( 4mM ) , or DMSO . Representative flow cytometer profiles are displayed . Average fractions of GFP + cells are calculated from duplicate assays . P1 - GFP , Prdm1 - GFP .

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FACS analysis of Prdm1-GFP positive (GFP+) cells in day-2 embryoids aggregated under addition of LIF (10ng ml-1) and BMP4 (500ng ml-1), dm-αKG (4mM), or DMSO. Representative flow cytometer profiles are displayed. Average fractions of GFP+ cells are calculated from duplicate assays. P1-GFP, Prdm1-GFP.


FACS analysis of Prdm1 - GFP positive ( GFP + ) cells in day - 4 PGCLC aggregates specified from 4mM dm - αKG - treated ( t = 48h to t = 72h ) EpiLCs . Representative flow cytometer profiles are depicted . The average fractions of GFP + cells , quantified from triplicate experiments , are shown . P1 - GFP , Prdm1 - GFP .

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FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 PGCLC aggregates specified from 4mM dm-αKG-treated (t=48h to t=72h) EpiLCs. Representative flow cytometer profiles are depicted. The average fractions of GFP+ cells, quantified from triplicate experiments, are shown. P1-GFP, Prdm1-GFP.


Transcript analysis by qRT - PCR of PGC specifiers , demethylating enzymes , mesoderm , endoderm , and ESC regulators in FACS - sorted day - 4 Prdm1 - GFP embryoids induced from 4mM dm - αKG - treated 72h EpiLCs . Expression levels are normalized to Prdm1 - GFP negative cells from control embryoids . Graphs represent averages from triplicate experiments . + , Prdm1 - GFP positive cells ; - , Prdm1 - GFP negative cells .

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Transcript analysis by qRT-PCR of PGC specifiers, demethylating enzymes, mesoderm, endoderm, and ESC regulators in FACS-sorted day-4 Prdm1-GFP embryoids induced from 4mM dm-αKG-treated 72h EpiLCs. Expression levels are normalized to Prdm1-GFP negative cells from control embryoids. Graphs represent averages from triplicate experiments. +, Prdm1-GFP positive cells; -, Prdm1-GFP negative cells.


Western blot analysis for H3K9me2 , H3K27me3 , and DNMT3b in 4mM dm - αKG - treated EpiLCs . H3 is used as a loading control . 48h , dm - αKG and DMSO , respectively , supplementation from t = 24h to t = 48h during the EpiLC differentiation . 72h , dm - αKG and DMSO , respectively , supplementation from t = 48h to t = 72h during the EpiLC differentiation .

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Western blot analysis for H3K9me2, H3K27me3, and DNMT3b in 4mM dm-αKG-treated EpiLCs. H3 is used as a loading control. 48h, dm-αKG and DMSO, respectively, supplementation from t=24h to t=48h during the EpiLC differentiation. 72h, dm-αKG and DMSO, respectively, supplementation from t=48h to t=72h during the EpiLC differentiation.


ChIP - qPCR analysis of H3K9me2 and H3K27me3 in putative enhancer regions of genes associated with the naïve pluripotent state ( Esrrb , Arid5b ) and PGC fate ( Tfap2c ) , respectively , in naïve ESCs and at t = 48h following EpiLC induction in the presence of 4mM dm - αKG and DMSO , respectively . Graphs show enrichment of H3K9me2 , H3K27me3 , and IgG control , respectively , relative to DMSO - treated EpiLCs .

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ChIP-qPCR analysis of H3K9me2 and H3K27me3 in putative enhancer regions of genes associated with the naïve pluripotent state (Esrrb, Arid5b) and PGC fate (Tfap2c), respectively, in naïve ESCs and at t=48h following EpiLC induction in the presence of 4mM dm-αKG and DMSO, respectively. Graphs show enrichment of H3K9me2, H3K27me3, and IgG control, respectively, relative to DMSO-treated EpiLCs.


( A ) SA - β - gal staining of U2 - OS cells , harvested at indicated times post irradiation ( IR ) ; h , hours ; D , days ; UT , untreated . n = 3 . Scale bar : 50 μm . Bar Graph : Significance was determined by Brown Forsythe ANOVA with Dunnett ' s 3T test , using n = 3 biological replicates . SD shown . * * * p < 0 . 001 , n . s . = not significant .

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(A) SA-β-gal staining of U2-OS cells, harvested at indicated times post irradiation (IR); h, hours; D, days; UT, untreated. n = 3. Scale bar: 50 μm. Bar Graph: Significance was determined by Brown Forsythe ANOVA with Dunnett's 3T test, using n = 3 biological replicates. SD shown. *** p < 0.001, n.s. = not significant.


( B ) Nuclear fractions from U2 - OS cells were harvested at indicated time points post IR ( 20 Gy ) as in ( A ) . Top panel : NF - κB DNA binding was analyzed by EMSA . Lower panels : Western blot of indicated NF - κB subunits and PARP1 as loading control . The two phases of nuclear

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(B) Nuclear fractions from U2-OS cells were harvested at indicated time points post IR (20 Gy) as in (A). Top panel: NF-κB DNA binding was analyzed by EMSA. Lower panels: Western blot of indicated NF-κB subunits and PARP1 as loading control. The two phases of nuclear translocation


( C ) Gene expression analysis of two NF - κB phases following damage . RNA - Seq analysis ( n = 5 biological replicates per group ) of U2 - OS cells , either untreated or analyzed 1 . 5 hours or 7 days after irradiation ( 20 Gy ) , as indicated . The heatmap shows significantly regulated genes ( log2 value > 0 . 5 and p value < 0 . 05 ) . Black arrow points to NFKBIA . Significance determined by ANOVA with Bonferroni correction for multiple testing . See also Dataset EV1 .

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(C) Gene expression analysis of two NF-κB phases following damage. RNA-Seq analysis (n = 5 biological replicates per group) of U2-OS cells, either untreated or analyzed 1.5 hours or 7 days after irradiation (20 Gy), as indicated. The heatmap shows significantly regulated genes (log2 value > 0.5 and p value < 0.05). Black arrow points to NFKBIA. Significance determined by ANOVA with Bonferroni correction for multiple testing. See also Dataset EV1.


( D ) Female mice 12 - 16 weeks old ( n = 6 mice per condition ( with three technical repeats per mouse ) , were irradiated ( 5 Gy ) and sacrificed after 1 hour ( first phase ) , 7 days ( second phase ) or left untreated . Skin or renal tissue RNA was analyzed by RT - qPCR , shown as a mean + / - SD . Significance was confirmed by ANOVA with Tukey multiple comparisons test . SD shown : * = p < 0 . 05 , * * = p < 0 . 01 .

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(D) Female mice 12-16 weeks old (n = 6 mice per condition (with three technical repeats per mouse), were irradiated (5 Gy) and sacrificed after 1 hour (first phase), 7 days (second phase) or left untreated. Skin or renal tissue RNA was analyzed by RT-qPCR, shown as a mean +/- SD. Significance was confirmed by ANOVA with Tukey multiple comparisons test. SD shown: * = p < 0.05, ** = p < 0.01.


( E ) In situ hybridization using an anti - sense IκBα / Nfkbia riboprobe on longitudinal skin sections from male and female mice ( 5 - 8 weeks of age ) treated as in ( D ) for the time indicated . Representative sections shown from n = 6 mice per condition . Bu , bulge region ( dashed lines ) , HS , hair shaft , IFE , interfollicular epidermis . Scale bar : 50 μm . ( F ) Quantitation of ( E ) from n = 6 mice per condition and 3 - 4 sections per mouse . Bulge regions positive for Nfkbia were counted and presented as percentage of total . One way ANOVA analysis with Tukey multiple comparisons test was performed . SD shown . * * = p < 0 . 01 .

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(E) In situ hybridization using an anti-sense IκBα/Nfkbia riboprobe on longitudinal skin sections from male and female mice (5-8 weeks of age) treated as in (D) for the time indicated. Representative sections shown from n = 6 mice per condition. Bu, bulge region (dashed lines), HS, hair shaft, IFE, interfollicular epidermis. Scale bar: 50 μm. (F) Quantitation of (E) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for Nfkbia were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. ** = p < 0.01.


( G ) Skin sections as in ( E ) analyzed by immunofluorescence with IκBα ( red ) or IL - 6 antibody ( green ) and nuclear DAPI staining ( blue ) . Dotted lines delineate hair follicles , Bu , bulge region , HS , hair shaft , IFE , interfollicular epidermis , SG , sebaceous gland . Scale bar : 50 μm . ( H ) Quantitation of ( G ) from n = 6 mice per condition and 3 - 4 sections per mouse . Bulge regions positive for IκBα or IL - 6 were counted and presented as percentage of total . One way ANOVA analysis with Tukey multiple comparisons test was performed . SD shown . * = p < 0 . 05 , * * * = p < 0 . 001 .

(G) Skin sections as in (E) analyzed by immunofluorescence with IκBα (red) or IL-6 antibody (green) and nuclear DAPI staining (blue). Dotted lines delineate hair follicles, Bu, bulge region, HS, hair shaft, IFE, interfollicular epidermis, SG, sebaceous gland. Scale bar: 50 μm. (H) Quantitation of (G) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for IκBα or IL-6 were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. * = p < 0.05, *** = p < 0.001.


( A ) Left panel : IκBα western blot of U2 - OS cells . Lane 1 , untreated ( ut ) ; Lanes 2 - 4 , single IR ( 20 Gy ) at indicated time points prior to harvest ; Lane 5 , IR at 5 days prior to harvest , plus 1 . 5 hours prior to harvest . Right panel : Samples treated as in the left panel , analyzed by RT - qPCR . Significance from n = 3 replicates per condition determined by ANOVA analysis with Tukey multiple comparisons test . SD shown . * * * = p < 0 . 001 .

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(A) Left panel: IκBα western blot of U2-OS cells. Lane 1, untreated (ut); Lanes 2-4, single IR (20 Gy) at indicated time points prior to harvest; Lane 5, IR at 5 days prior to harvest, plus 1.5 hours prior to harvest. Right panel: Samples treated as in the left panel, analyzed by RT-qPCR. Significance from n = 3 replicates per condition determined by ANOVA analysis with Tukey multiple comparisons test. SD shown. *** = p < 0.001.


( B ) NFKBIA knockdown by dox - inducible shRNA in U2 - OS cells . Cells were pre - treated with Dox to induce knockdown . For knockdown efficiency see Fig . S2F . Cells were irradiated ( 20 Gy ) or not , as indicated and described above . NF - κB activity was analyzed by EMSA . ns , non - specific band . A representative gel from n = 3 biological replicates is shown .

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(B) NFKBIA knockdown by dox-inducible shRNA in U2-OS cells. Cells were pre-treated with Dox to induce knockdown. For knockdown efficiency see Fig. S2F. Cells were irradiated (20 Gy) or not, as indicated and described above. NF-κB activity was analyzed by EMSA. ns, non-specific band. A representative gel from n = 3 biological replicates is shown.


( E ) GSEA was performed comparing the IκBα IEC ‐ KO gene list ( n = 4 villin - Cre x floxed IκBα mice and n = 4 littermate controls ( GSE 139251 ) Mikuda et al , 2020 ( ) , with a gene signature based on a list of SASP factors Coppe et al , 2010 ( ) . NES : normalized enrichment score . FDR : false discovery rate .

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(E) GSEA was performed comparing the IκBα IEC‐KO gene list (n = 4 villin-Cre x floxed IκBα mice and n = 4 littermate controls (GSE 139251) Mikuda et al, 2020(), with a gene signature based on a list of SASP factors Coppe et al, 2010(). NES: normalized enrichment score. FDR: false discovery rate.


( F ) RT - qPCR analysis of indicated genes was performed using RNA extracted from the duodenum of 5 and 8 weeks old control villin - Cre ( n = 6 ) or villin - Cre x floxed IκBα mice ( n = 6 ) both male and female . Expression is shown as fold change between littermates , paired t - test . SD shown . * p < 0 . 05 * * = P < 0 . 01 .

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(F) RT-qPCR analysis of indicated genes was performed using RNA extracted from the duodenum of 5 and 8 weeks old control villin-Cre (n = 6) or villin-Cre x floxed IκBα mice (n = 6) both male and female. Expression is shown as fold change between littermates, paired t-test. SD shown. * p < 0.05 ** = P < 0.01.


( A ) Nuclear fractions from either untreated or irradiated ( 20 Gy ) U2 - OS cells , 1 . 5 hours or 7 days prior to harvest , were analyzed by EMSA with supershift analysis . For supershifting , lysates were incubated with antibodies or water , as indicated . Arrows point to shift location of a given subunit , due to antibody binding . ns , non - specific band . All antibodies were previously tested for supershift compatibility . Representative gel shown from n = 3 replicates .

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(A) Nuclear fractions from either untreated or irradiated (20 Gy) U2-OS cells, 1.5 hours or 7 days prior to harvest, were analyzed by EMSA with supershift analysis. For supershifting, lysates were incubated with antibodies or water, as indicated. Arrows point to shift location of a given subunit, due to antibody binding. ns, non-specific band. All antibodies were previously tested for supershift compatibility. Representative gel shown from n = 3 replicates.


( B ) Expression of SASP targets of NF - κB obtained from RNA - seq analysis ( Dataset EV1B ) , was quantitated using RT - qPCR from the shRNA ( RELA ) stably expressing U2 - OS cells . For knockdown efficiency see Appendix Fig S4A . Cells were treated with doxycycline ( Dox ) to induce knockdown of p65 . Heatmap represents targets normalized to the untreated Scrambled control . Expression is shown as log2 change with p values < 0 . 05 . Statistical analysis performed using ANOVA with Bonferroni correction for multiple testing . First - phase and second - phase samples were irradiated ( 20 Gy ) 1 . 5h and 7 Days prior to harvest , respectively . Analysis based on n = 3 biological replicates .

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(B) Expression of SASP targets of NF-κB obtained from RNA-seq analysis (Dataset EV1B), was quantitated using RT-qPCR from the shRNA (RELA) stably expressing U2-OS cells. For knockdown efficiency see Appendix Fig S4A. Cells were treated with doxycycline (Dox) to induce knockdown of p65. Heatmap represents targets normalized to the untreated Scrambled control. Expression is shown as log2 change with p values < 0.05. Statistical analysis performed using ANOVA with Bonferroni correction for multiple testing. First-phase and second-phase samples were irradiated (20 Gy) 1.5h and 7 Days prior to harvest, respectively. Analysis based on n = 3 biological replicates.


( C ) Cell duplication was measured at time points indicated in U2 - OS cells bearing either scrambled control or Dox - inducible shRNA against RELA ( n = 3 biological replicates ) . Treatment with Dox was initiated at two days prior to IR ( 20 Gy ) or after IR . Statistical significance in total duplication number between groups at day 6 was determined by ANOVA with Tukey multiple comparisons test . SD shown . * = P < 0 . 05 , * * * = p < 0 . 001 .

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(C) Cell duplication was measured at time points indicated in U2-OS cells bearing either scrambled control or Dox-inducible shRNA against RELA (n = 3 biological replicates). Treatment with Dox was initiated at two days prior to IR (20 Gy) or after IR. Statistical significance in total duplication number between groups at day 6 was determined by ANOVA with Tukey multiple comparisons test. SD shown. * = P < 0.05, *** = p < 0.001.


( A ) Nuclear ( Nuc . ) and cytoplasmic ( Cyto . ) fractions of U2 - OS cells were analyzed at times indicated after IR exposure by SDS - PAGE western blotting . PARP1 and LDHA are fractionation controls and phospho - specific p65 signals are indicated . Representative gels from n = 3 biological replicates are shown . Right panel : quantitation of nuclear p65 and phosphorylated p65 species ( n = 3 ) . Fold changes compared to untreated samples ( ut ) and time points post IR are indicated on Y and X axes , respectively .

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(A) Nuclear (Nuc.) and cytoplasmic (Cyto.) fractions of U2-OS cells were analyzed at times indicated after IR exposure by SDS-PAGE western blotting. PARP1 and LDHA are fractionation controls and phospho-specific p65 signals are indicated. Representative gels from n = 3 biological replicates are shown. Right panel: quantitation of nuclear p65 and phosphorylated p65 species (n = 3). Fold changes compared to untreated samples (ut) and time points post IR are indicated on Y and X axes, respectively.


( B ) U2 - OS cells treated with dox to deplete endogenous p65 , were irradiated ( 20 Gy ) and transfected with plasmids encoding p65 , p65 - S276A or p65 - S468A , as indicated . Nuclear ( N ) and cytoplasmic ( C ) lysates were analyzed by SDS - PAGE at day seven post IR . Representative gel from n = 3 biological replicates is shown . PARP1 and αTubulin serve as fractionation and loading controls .

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(B) U2-OS cells treated with dox to deplete endogenous p65, were irradiated (20 Gy) and transfected with plasmids encoding p65, p65-S276A or p65-S468A, as indicated. Nuclear (N) and cytoplasmic (C) lysates were analyzed by SDS-PAGE at day seven post IR. Representative gel from n = 3 biological replicates is shown. PARP1 and αTubulin serve as fractionation and loading controls.