Quantification of Ki67 immunohistochemistry staining of Eso26 xenograft tumors at an early timepoint ( 5 days ) or post treatment / late timepoint ( > 22 days ) after treatment initiation with APR - 246 ( 50mg / kg ) + / - MK - 571 ( 50mg / kg ) . Dots indicate individual tumors .

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

Quantification of Ki67 immunohistochemistry staining of Eso26 xenograft tumors at an early timepoint (5 days) or post treatment / late timepoint (>22 days) after treatment initiation with APR-246 (50mg/kg) +/- MK-571 (50mg/kg). Dots indicate individual tumors.


Quantification of cleaved caspase 3 immunohistochemistry staining of Eso26 xenograft tumors in same tumors as shown in Fig . 2C . Dots indicate individual tumors .

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

Quantification of cleaved caspase 3 immunohistochemistry staining of Eso26 xenograft tumors in same tumors as shown in Fig. 2C. Dots indicate individual tumors.


Representative images of hematoxylin / eosin ( H & E ) staining and immunostaining of Ki67 , cleaved caspase 3 and p53 , post treatment / late timepoint ( > 22 days after treatment initiation ) with APR - 246 ( 50mg / kg ) + / - MK - 571 ( 50mg / kg ) .

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

Representative images of hematoxylin/eosin (H&E) staining and immunostaining of Ki67, cleaved caspase 3 and p53, post treatment / late timepoint (>22 days after treatment initiation) with APR-246 (50mg/kg) +/- MK-571 (50mg/kg).


Growth suppression determined by the ATP - based CTG assay in colorectal cancer patient - derived organoids ( colo - PDO ) after treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Growth suppression determined by the ATP-based CTG assay in colorectal cancer patient-derived organoids (colo-PDO) after treatment with APR-246 +/- MK-571 (n = 3).


IC50 values ( µM ) APR - 246 + / - 20 μM MK - 571 in indicated PDOs . * p = 0 . 04 , paired t test ( n = 3 except colo - PDO2 and eso - PDO3 where n = 2 ) .

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

IC50 values (µM) APR-246 +/- 20 μM MK-571 in indicated PDOs. *p = 0.04, paired t test (n = 3 except colo-PDO2 and eso-PDO3 where n = 2).


Organoid area ( in black , left y - axis ) and PI intensity ( in red , right y - axis ) of colo - PDO1 and colo - PDO2 as determined by image analysis 72h after treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Organoid area (in black, left y-axis) and PI intensity (in red, right y-axis) of colo-PDO1 and colo-PDO2 as determined by image analysis 72h after treatment with APR-246 +/- MK-571 (n = 3).


14C accumulation ( cpm / mg x ml - 1 ) in 11 cell lines after 24h 14C - APR - 246 + / - MK - 571 treatment ( n ≥ 3 ) . * p = 0 . 0006 , paired t - test . 14C - accumulation and n of individual cell lines as shown in Table S3 .

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

14C accumulation (cpm/mg x ml-1) in 11 cell lines after 24h 14C -APR-246 +/- MK-571 treatment (n ≥ 3). *p = 0.0006, paired t-test. 14C-accumulation and n of individual cell lines as shown in Table S3.


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 ( WT and R248W ) cells after 24h + treatment with 14C - APR - 246 + / - MK - 571 or Reversan ( n ≥ 3 , each dots in figure ) .

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

14C accumulation (cpm/mg x ml-1) in HCT116 (WT and R248W) cells after 24h +treatment with 14C -APR-246 +/- MK-571 or Reversan (n ≥ 3, each dots in figure).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT and R248W cells ( n ≥ 3 and ≥ 2 ) transfected with MRP1 siRNAs after 24h of 14C - APR - 246 treatment . Indicated values represent four individual MRP1 siRNAs and two individual negative control ( Ctrl ) siRNAs , where each dot is an individual siRNA . Mean of 14C - accumulation after treatment and n with individual siRNA sequences is shown in Table S4 . * p = 0 . 04 and for R248W p = 0 . 06 , Wilcoxon matched pairs signed rank test .

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

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells (n ≥ 3 and ≥ 2) transfected with MRP1 siRNAs after 24h of 14C-APR-246 treatment. Indicated values represent four individual MRP1 siRNAs and two individual negative control (Ctrl) siRNAs, where each dot is an individual siRNA. Mean of 14C-accumulation after treatment and n with individual siRNA sequences is shown in Table S4. *p = 0.04 and for R248W p = 0.06, Wilcoxon matched pairs signed rank test.


APR - 246 content in OVCAR - 3 cells ( TP53 R248Q ) 24h after treatment with APR - 246 + / - MK - 571 , ( n = 3 ) . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 .

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

APR-246 content in OVCAR-3 cells (TP53 R248Q) 24h after treatment with APR-246 +/- MK-571, (n = 3). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Glutathione - conjugated MQ ( GS - MQ ) in OVCAR - 3 cells after 24h treatment with APR - 246 + / - MK - 571 ( n = 2 ) . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 .

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

Glutathione-conjugated MQ (GS-MQ) in OVCAR-3 cells after 24h treatment with APR-246 +/- MK-571 (n = 2). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Amount of GS - MQ at indicated timepoints after incubation of GS - MQ + / - NAC ( n = 2 ) . See Fig . EV4 for chemical reaction . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 .

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

Amount of GS-MQ at indicated timepoints after incubation of GS-MQ +/- NAC (n = 2). See Fig. EV4 for chemical reaction. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Amount of GS - MQ by LC - MS ( green line , left axis ) and NAC - MQ ( purple line , right axis ) over time after incubation of GS - MQ with NAC ( n = 2 ) . Values on y - axes are not comparable because GS - MQ and NAC - MQ have different response signals on MS . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 .

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

Amount of GS-MQ by LC-MS (green line, left axis) and NAC-MQ (purple line, right axis) over time after incubation of GS-MQ with NAC (n = 2). Values on y-axes are not comparable because GS-MQ and NAC-MQ have different response signals on MS. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Mean IC50 ( µM ) values of APR - 246 in 17 different cell lines ( n ≥ 3 ) as shown by WST - 1 . * p = 0 . 03 , Mann Whitney test . Mean IC50 ( µM ) values for APR - 246 treatment and n for individual cell lines are also shown in Fig 1C and listed in Table S1 . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Mean IC50 (µM) values of APR-246 in 17 different cell lines (n ≥ 3) as shown by WST-1. *p = 0.03, Mann Whitney test. Mean IC50 (µM) values for APR-246 treatment and n for individual cell lines are also shown in Fig 1C and listed in Table S1. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Box - and - whisker plot of Pearson correlations between PRIMA - 1 area - under - the - curve ( AUC ) and the levels of 225 different metabolites from the DepMap portal . Central band indicates median , boxes indicate 25th and 75th percentile , and whiskers show 2 . 5th and 97 . 5th percentile outlier metabolites . High GSH and GSSG ( in red ) correlate with low PRIMA - 1 sensitivity . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Box-and-whisker plot of Pearson correlations between PRIMA-1 area-under-the-curve (AUC) and the levels of 225 different metabolites from the DepMap portal. Central band indicates median, boxes indicate 25th and 75th percentile, and whiskers show 2.5th and 97.5th percentile outlier metabolites. High GSH and GSSG (in red) correlate with low PRIMA-1 sensitivity. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG ) as shown by glutathione reductase ( GR ) re - cycling assay in H1299 R175H ( tet - off ) ( + / - doxycycline ) and H1299 - / - cells 24h after seeding ( n = 3 ) . Indicated values are fold change compared to mutant TP53 cells . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Total intracellular glutathione (GSH+GSSG) as shown by glutathione reductase (GR) re-cycling assay in H1299 R175H (tet-off) (+/- doxycycline) and H1299 -/- cells 24h after seeding (n = 3). Indicated values are fold change compared to mutant TP53 cells. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG ) as shown by GR re - cycling assay in untreated HCT116 ( R248W , WT and - / - ) cells 48h after seeding ( n = 3 , 5 or 2 respectively ) . Indicated values are fold change to mutant TP53 cells . Part of this data is shown in Fig . EV4 . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Total intracellular glutathione (GSH+GSSG) as shown by GR re-cycling assay in untreated HCT116 (R248W, WT and -/-) cells 48h after seeding (n = 3, 5 or 2 respectively). Indicated values are fold change to mutant TP53 cells. Part of this data is shown in Fig. EV4. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells ( n = 2 ) Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells (n = 2) Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Correlation between 14C accumulation ( cpm / mg x ml - 1 ) after treatment with 25 µM 14C - APR - 246 and IC50 ( µM ) values for APR - 246 in 11 cell lines ( n ≥ 3 ) ( r = - 0 . 66 , p = 0 . 03 , Pearson correlation ) . Each dot represents one cell line , 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3 . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Correlation between 14C accumulation (cpm/mg x ml-1) after treatment with 25 µM 14C-APR-246 and IC50 (µM) values for APR-246 in 11 cell lines (n ≥ 3) (r = -0.66, p = 0.03, Pearson correlation). Each dot represents one cell line, 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


ABCC1 mRNA Expression , RSEM ( Batch normalized from Illumina HiSeq _ RNASeqV2 ) in lung cancer ( luad & lusc studies , * p < 0 . 0001 ) and colon adenocarcinoma ( coad study , * p = 0 . 005 ) grouped into having no alterations or putative driver missense mutations in TP53 . Statistical analysis by Mann Whitney test , n is indicated in the figure . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

ABCC1 mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) in lung cancer (luad & lusc studies, *p<0.0001) and colon adenocarcinoma (coad study, *p=0.005) grouped into having no alterations or putative driver missense mutations in TP53. Statistical analysis by Mann Whitney test, n is indicated in the figure. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


14C accumulation ( cpm / mg x ml - 1 ) in H1299 R175H cells ( + / - doxycycline ) and H1299 - / - cells after 24h treatment with 14C - APR - 246 ( n = 2 ) . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

14C accumulation (cpm/mg x ml-1) in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells after 24h treatment with 14C-APR-246 (n = 2). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Growth suppression in H1299 R175H cells ( + / - doxycycline ) and H1299 - / - cells treated for 72h with APR - 246 as shown by WST - 1 assay ( n = 3 ) . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Growth suppression in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells treated for 72h with APR-246 as shown by WST-1 assay (n = 3). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG per 106 cells ) , as determined by GR re - cycling assay after 24h MK - 571 treatment in six different cell lines ( n ≥ 3 , except HCT116 WT n = 2 ) . * p = 0 . 014 , Paired t test . Each dot represents one cell line , see Fig . EV6A for individual cell lines and n .

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

Total intracellular glutathione (GSH + GSSG per 106 cells), as determined by GR re-cycling assay after 24h MK-571 treatment in six different cell lines (n ≥ 3, except HCT116 WT n = 2). *p = 0.014, Paired t test. Each dot represents one cell line, see Fig. EV6A for individual cell lines and n.


Intracellular oxidized glutathione ( GSSG per 106 cells ) as shown by LC - MS in OVCAR - 3 ( R248Q TP53 ) cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Intracellular reduced glutathione ( GSH per 106 cells ) as shown by LC - MS in OVCAR - 3 cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Intracellular reduced glutathione (GSH per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Western blot analysis of MRP1 ( Cell Signaling ) , xCT and GAPDH of OVCAR - 3 cells treated with APR - 246 + / - MK - 571 for 24h .

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

Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of OVCAR-3 cells treated with APR-246 +/- MK-571 for 24h.


Western blot analysis of MRP1 ( Cell Signaling ) , xCT and GAPDH of HCT116 WT and R248W cells treated with APR - 246 + / - MK - 571 for 24h .

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

Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24h.


Intracellular cystine ( CySS per 106 cells ) as shown by LC - MS in OVCAR - 3 cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Intracellular cystine (CySS per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Intracellular cysteine ( Cys per 106 cells ) as shown by LC - MS in OVCAR - 3 cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

Intracellular cysteine (Cys per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT and R248W mutant TP53 cells + - transfected with MRP1 or xCT siRNA following 24h of 14C - APR - 246 treatment + / - MK - 571 . Indicated values are mean values from four MRP1 siRNAs ( n ≥ 3 ) , three xCT siRNAs ( n = 1 - 2 ) and two control siRNAs ( n ≥ 3 ) . Mean 14C - accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C - APR - 246 treatment is also shown in Fig 3D .

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

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W mutant TP53 cells +-transfected with MRP1 or xCT siRNA following 24h of 14C -APR-246 treatment +/- MK-571 . Indicated values are mean values from four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n = 1-2) and two control siRNAs (n ≥ 3). Mean 14C -accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C -APR-246 treatment is also shown in Fig 3D.


Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR - 246 treatment as shown by WST - 1 assay . Indicated values are mean values of four MRP1 siRNAs ( n ≥ 3 ) , three xCT siRNAs ( n = 1 - 2 ) and two control siRNAs ( n ≥ 3 ) . Data for control and MRP1 siRNA after APR - 246 single treatment are also included in Fig 1I . Values and n for individual siRNA shown in Fig . S7C .

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

Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR-246 treatment as shown by WST-1 assay. Indicated values are mean values of four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n =1-2) and two control siRNAs (n ≥ 3). Data for control and MRP1 siRNA after APR-246 single treatment are also included in Fig 1I. Values and n for individual siRNA shown in Fig. S7C.


Total intracellular glutathione ( GSH + GSSG per 106 cells ) in HCT116 WT cells as shown by GR re - cycling assay 48h post transfection of siRNA . Indicated values are means from four MRP1 siRNAs ( n = 3 ) , two xCT siRNA ( n = 2 - 3 ) and two control siRNA ( n = 2 - 3 ) .

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Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT cells as shown by GR re-cycling assay 48h post transfection of siRNA. Indicated values are means from four MRP1 siRNAs (n = 3), two xCT siRNA (n = 2-3) and two control siRNA (n = 2-3).


Total intracellular glutathione ( GSH + GSSG per 106 cells ) in HCT116 WT and R248W cells measured by GR re - cycling assay after 24h of MK - 571 or sulfasalazine ( SSZ ) treatment ( n ≥ 3 , except SSZ where n = 2 , n indicated by dots ) .

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Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT and R248W cells measured by GR re-cycling assay after 24h of MK-571 or sulfasalazine (SSZ) treatment (n ≥ 3, except SSZ where n = 2, n indicated by dots).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT and R248W cells at 24h treatment with 14C - APR - 246 , combined with MK - 571 or sulfasalazine ( SSZ ) , ( n ≥ 4 , except SSZ where n = 2 , n indicated by dots ) . Data for 14C - APR - 246 + / - MK - 571 is also shown in Fig 3C and S3D .

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14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells at 24h treatment with 14C -APR-246, combined with MK-571 or sulfasalazine (SSZ), (n ≥ 4, except SSZ where n = 2, n indicated by dots). Data for 14C -APR-246 +/- MK-571 is also shown in Fig 3C and S3D.


Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR - 246 + / - MK - 571 or sulfasalazine ( SSZ ) , or untreated ( n ≥ 4 , except SSZ where n = 2 , n indicated by dots )

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Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ), or untreated (n ≥ 4, except SSZ where n = 2, n indicated by dots)


OVCAR - 3 ( R248Q TP53 ) confluence by IncuCyte ® during 72h treatment with APR - 246 + / - MK - 571 or sulfasalzine ( SSZ ) ( n = 3 ) . Part of the data are shown in Fig 1G . Growth suppression in OVCAR - 3 cells as determined by IncuCyte ® during 72h treatment with APR - 246 + / - MK - 571 or sulfasalazine ( SSZ ) depicted as heatmaps ( n = 3 ) .

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OVCAR-3 (R248Q TP53) confluence by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalzine (SSZ) (n = 3). Part of the data are shown in Fig 1G. Growth suppression in OVCAR-3 cells as determined by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ) depicted as heatmaps (n = 3).


a , The 2 - bp deletion identified in exon 9b of patient 1 . b , The T - to - A nonsense mutation in exon 4 of patient 2 ( stop codon is underlined ) . c , Upper panel , intronic mutation in patient 3 ; lower panel , sequencing of the RT - PCR product in patient 3 , showing exon - 6 skipping . d , Upper panel , The G - to - A point mutation at the splice - donor site in intron 5 of patient 6 ; lower panel , sequencing of the RT - PCR product in patient 6 , showing a 6 - bp insertion at the junction between exons 5 and 6 . The inserted sequence ( underlined ) is derived from the first six nucleotides of intron 5 ( underlined ) , which creates a stop codon ( dotted ) . Most probably , nucleotides 7 and 8 , GT ( double underlined ) , in intron 5 are alternatively recognized as a splice - donor site . e , Upper panel , the 10 - bp deletion in patient 9 . This mutation deletes four nucleotides from intron 5 and six from exon 6 . Lower panel , sequence of the RT - PCR product in patient 9 reveals the deletion of the 10 nucleotides at the 5 ′ end of exon 6 . The ' AG ' ( double underlined ) in exon 6 immediately after the genomic DNA deletion appears to be recognized as an alternative splice - acceptor site .

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a, The 2-bp deletion identified in exon 9b of patient 1. b, The T-to-A nonsense mutation in exon 4 of patient 2 (stop codon is underlined). c, Upper panel, intronic mutation in patient 3; lower panel, sequencing of the RT-PCR product in patient 3, showing exon-6 skipping. d, Upper panel, The G-to-A point mutation at the splice-donor site in intron 5 of patient 6; lower panel, sequencing of the RT-PCR product in patient 6, showing a 6-bp insertion at the junction between exons 5 and 6. The inserted sequence (underlined) is derived from the first six nucleotides of intron 5 (underlined), which creates a stop codon (dotted). Most probably, nucleotides 7 and 8, GT (double underlined), in intron 5 are alternatively recognized as a splice-donor site. e, Upper panel, the 10-bp deletion in patient 9. This mutation deletes four nucleotides from intron 5 and six from exon 6. Lower panel, sequence of the RT-PCR product in patient 9 reveals the deletion of the 10 nucleotides at the 5′ end of exon 6. The 'AG' (double underlined) in exon 6 immediately after the genomic DNA deletion appears to be recognized as an alternative splice-acceptor site.


Figure 2 Representation of the mutant LAMP - 2b . ' Normal ' represents the LAMP - 2b amino - acid sequence . Because patient 1 , who developed full DD symptoms , has a mutation in exon 9b , the LAMP - 2b isoform is most probably responsible for DD . Residue 265 , one of the putative disulphide - bond - creating cysteines , and two glycosylation sites are lost by exon - 6 skipping in patient 3 , which probably results in a significant change in the secondary structure . Black boxes represent the loops created between two cystein residues flanking each of the four loop regions ; Y depicts a glycosylation site ; hatched boxes show altered amino acids caused by frameshift mutations ; dashes represent missing amino acids due to exon - 6 skipping in patient 3 . All of the other mutant proteins lack the transmembrane domain ( grey bar ) because of nonsense or frameshift mutations . Thus , those molecules cannot function as lysosomal ' membrane ' proteins .

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Figure 2 Representation of the mutant LAMP-2b. 'Normal' represents the LAMP-2b amino-acid sequence. Because patient 1, who developed full DD symptoms, has a mutation in exon 9b, the LAMP-2b isoform is most probably responsible for DD. Residue 265, one of the putative disulphide-bond-creating cysteines, and two glycosylation sites are lost by exon-6 skipping in patient 3, which probably results in a significant change in the secondary structure. Black boxes represent the loops created between two cystein residues flanking each of the four loop regions; Y depicts a glycosylation site; hatched boxes show altered amino acids caused by frameshift mutations; dashes represent missing amino acids due to exon-6 skipping in patient 3. All of the other mutant proteins lack the transmembrane domain (grey bar) because of nonsense or frameshift mutations. Thus, those molecules cannot function as lysosomal 'membrane' proteins.


a , Skeletal muscle . Muscle extracts from patients 1 - 6 and 10 and a control were blotted and labelled with antibodies against LAMP - 2 ( top panel ) and β - actin ( bottom panel ) . LAMP - 2 is deficient in all muscles examined except the sample from patient 1 that showed a trace amount of protein , which is probably the LAMP - 2a isoform . In support of this idea , LAMP - 2b is the principal LAMP - 2 species in skeletal muscle6 . Numbers denote patients as in Table 1 ; C , control . b , Heart . Protein was extracted from cardiac muscle of patient 4 and a control , blotted onto a nitrocellulose membrane , and labelled with the antibodies against LAMP - 2 ( too panel ) and β - actin ( bottom panel ) . LAMP - 2 is deficient also in cardiac muscle . C , control ; 4 , patient 4 .

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a, Skeletal muscle. Muscle extracts from patients 1-6 and 10 and a control were blotted and labelled with antibodies against LAMP-2 (top panel) and β-actin (bottom panel). LAMP-2 is deficient in all muscles examined except the sample from patient 1 that showed a trace amount of protein, which is probably the LAMP-2a isoform. In support of this idea, LAMP-2b is the principal LAMP-2 species in skeletal muscle6. Numbers denote patients as in Table 1; C, control. b, Heart. Protein was extracted from cardiac muscle of patient 4 and a control, blotted onto a nitrocellulose membrane, and labelled with the antibodies against LAMP-2 (too panel) and β-actin (bottom panel). LAMP-2 is deficient also in cardiac muscle. C, control; 4, patient 4.


Serial transverse sections of the biopsiedmuscle from six DD patients , and samples from a XMEA patient and a normal control , were immunostained with antibodies against LAMP - 2 and limp - I . LAMP - 2 is absent in all DD muscles ( patient 6 shown here ) , whereas it is present in the XMEA muscle . The antibody against limp - I highlighted vacuoles in both DD and XMEA muscles . LAMP - 1 antibody faintly stained both DD and XMEA muscles , suggesting that the expression level of LAMP - 1 might be lower in skeletal muscle than LAMP - 2 or limp - I ( data not shown ) . The muscle from the patient without a lamp - 2 mutation also showed LAMP - 2 deficiency on immunohistochemistry similar to genetically proven DD patients with primary LAMP - 2 deficiency ( data not shown ) . The immunohistochemical stains of LAMP - 2 , limp - I , and LAMP - 1 in normal muscle shows numerous tiny particles and accentuates type 1 fibres . H & amp ; amp ; E , haematoxylin and eosin , original magnification × 150 .

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Serial transverse sections of the biopsiedmuscle from six DD patients, and samples from a XMEA patient and a normal control, were immunostained with antibodies against LAMP-2 and limp-I. LAMP-2 is absent in all DD muscles (patient 6 shown here), whereas it is present in the XMEA muscle. The antibody against limp-I highlighted vacuoles in both DD and XMEA muscles. LAMP-1 antibody faintly stained both DD and XMEA muscles, suggesting that the expression level of LAMP-1 might be lower in skeletal muscle than LAMP-2 or limp-I (data not shown). The muscle from the patient without a lamp-2 mutation also showed LAMP-2 deficiency on immunohistochemistry similar to genetically proven DD patients with primary LAMP-2 deficiency (data not shown). The immunohistochemical stains of LAMP-2, limp-I, and LAMP-1 in normal muscle shows numerous tiny particles and accentuates type 1 fibres. H&amp;E, haematoxylin and eosin, original magnification ×150.


( B ) Representative immunofluorescence images of HeLa cells in various mitotic stages . Cells were extracted , fixed , and co - stained for Cyclin B2 ( green ) , ACA ( red ) , and DNA ( blue ) . Scale bar , 10 µm .

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(B) Representative immunofluorescence images of HeLa cells in various mitotic stages. Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue). Scale bar, 10 µm.


( C ) Representative immunofluorescence images of HeLa cells that were fixed and co - stained for Cyclin B2 ( green ) , mAb414 ( red ) , and DNA ( blue ) . Scale bar , 10 µm .

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(C) Representative immunofluorescence images of HeLa cells that were fixed and co-stained for Cyclin B2 (green), mAb414 (red), and DNA (blue). Scale bar, 10 µm.


Representative immunofluorescence images of HeLa cells treated with an indicated siRNA Cells were extracted , fixed , and co - stained for Cyclin B2 ( green ) , ACA ( red ) , and DNA ( blue ) .

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Representative immunofluorescence images of HeLa cells treated with an indicated siRNA Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue).


Representative immunofluorescence images of HeLa cells treated with an indicated various drugs ( E ) . Cells were extracted , fixed , and co - stained for Cyclin B2 ( green ) , ACA ( red ) , and DNA ( blue ) .

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Representative immunofluorescence images of HeLa cells treated with an indicated various drugs (E). Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue).


( F ) HeLa cells were synchronized by double thymidine , then released and collected at selected time points . Cell lysate samples were resolved by SDS - PAGE and analyzed by Western blotting using anti - Cyclin B1 antibody , anti - Cyclin B2 antibody , or anti - β - Actin antibody .

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(F) HeLa cells were synchronized by double thymidine, then released and collected at selected time points. Cell lysate samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B1 antibody, anti-Cyclin B2 antibody, or anti-β-Actin antibody.


( B ) Immunoprecipitation assay performed with two different rabbit Cyclin B2 antibodies . Nocodazole - arrested mitotic cells were collected and lysed . Immunoprecipitation was accomplished using control IgG , anti - Cyclin B2 - 1 ( Abcam , ab185622 ) , and anti - Cyclin B2 - 2 ( Proteintech , 21644 - 1 - AP ) . Immunoprecipitation samples were resolved by Western blotting using a mouse anti - Cyclin B2 antibody ( Santa Cruz , sc - 28303 ) and a mouse anti - Mad2 antibody ( Santa Cruz , sc - 47747 )

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(B) Immunoprecipitation assay performed with two different rabbit Cyclin B2 antibodies. Nocodazole-arrested mitotic cells were collected and lysed. Immunoprecipitation was accomplished using control IgG, anti-Cyclin B2-1 (Abcam, ab185622), and anti-Cyclin B2-2 (Proteintech, 21644-1-AP). Immunoprecipitation samples were resolved by Western blotting using a mouse anti-Cyclin B2 antibody ( Santa Cruz, sc-28303) and a mouse anti-Mad2 antibody (Santa Cruz, sc-47747)


( C and D ) Flag immunoprecipitation assay . 293T cells were co - transfected with Flag - C - Mad2 and GFP - Cyclin B1 / B2 ( C ) , and with Flag - Mad1 - 1 - 215 and GFP - Cyclin B1 / B2 ( D ) . After 36 h , the cells were collected and lysed , and immunoprecipitation was accomplished with anti - Flag M2 beads . Immunoprecipitates were resolved by SDS - PAGE and analyzed by Western blotting using anti - Flag antibody and anti - GFP

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(C and D) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-C-Mad2 and GFP-Cyclin B1/B2 (C), and with Flag-Mad1-1-215 and GFP-Cyclin B1/B2 (D). After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-GFP antibody


( E ) GST pulldown assay . Purified GST , GST - Cyclin B1 - 165 - 433 , and GST - Cyclin B2 - 130 - 398 - bound agarose beads were used as affinity matrices to absorb purified Trx - 6 × His - tagged C - Mad2 recombinant protein . Absorbed proteins were analyzed by SDS - PAGE and Coomassie Brilliant Blue

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(E) GST pulldown assay. Purified GST, GST-Cyclin B1-165-433, and GST-Cyclin B2-130-398-bound agarose beads were used as affinity matrices to absorb purified Trx-6×His-tagged C-Mad2 recombinant protein. Absorbed proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining


( F - I ) Representative immunofluorescence images of HeLa cells stably expressing GFP - Cyclin B2 ( F ) or GFP - Cyclin B1 ( H ) . The cells were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h . Then cells were fixed and co - stained for Mad2 ( red ) and DNA ( blue ) . Scale bar , 10 µm . Scatter graphs illustrating kinetochore intensity of GFP - Cyclin B2 ( G ) or GFP - Cyclin B1 ( I ) in cells treated as in F and H , respectively . Bars represent the mean kinetochore intensity ( ± SD ) normalized to values of the siControl group . Each dot represents one cell ( ≥ 30 cells from three independent experiments )

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(F-I) Representative immunofluorescence images of HeLa cells stably expressing GFP-Cyclin B2 (F) or GFP-Cyclin B1 (H). The cells were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h. Then cells were fixed and co-stained for Mad2 (red) and DNA (blue). Scale bar, 10 µm. Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2 (G) or GFP-Cyclin B1 (I) in cells treated as in F and H, respectively. Bars represent the mean kinetochore intensity (±SD) normalized to values of the siControl group. Each dot represents one cell (≥30 cells from three independent experiments)


( B ) Flag immunoprecipitation assay . Flag - tagged Cyclin B2 - WT , Cyclin B2 - 4A , or Cyclin B1 were expressed in 293T cells and purified with anti - Flag M2 beads . Subsequently , the beads were incubated with mitotic HeLa lysates for 2 h , and , following extensive washes , immunoprecipitatates were resolved by SDS - PAGE and analyzed by Western blotting using anti - Flag antibody and anti - Mad2 antibody .

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(B) Flag immunoprecipitation assay. Flag-tagged Cyclin B2-WT, Cyclin B2-4A, or Cyclin B1 were expressed in 293T cells and purified with anti-Flag M2 beads. Subsequently, the beads were incubated with mitotic HeLa lysates for 2 h, and, following extensive washes, immunoprecipitatates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-Mad2 antibody.


( C ) Flag immunoprecipitation assay . 293T cells were co - transfected with Flag - Cyclin B2 - WT or Flag - Cyclin B2 - 4A together with GFP ( negative control ) and GFP - CDK1 . After 36 h , the cells were collected and lysed , and immunoprecipitation was accomplished with anti - Flag M2 beads . Immunoprecipitation samples were resolved by SDS - PAGE and analyzed by Western blotting using anti - GFP antibody and anti - Flag antibody .

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(C) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-Cyclin B2-WT or Flag-Cyclin B2-4A together with GFP (negative control) and GFP-CDK1. After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitation samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-GFP antibody and anti-Flag antibody.


( D ) Representative immunofluorescence images of HeLa cells transfected with GFP - Cyclin B2 - WT or GFP - Cyclin B2 - 4A . After 36 h of transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained for ACA ( red ) and DNA ( blue ) . ( E ) Scatter graphs illustrating kinetochore intensity of GFP - Cyclin B2 - WT / 4A treated as in D . Bars represent the mean kinetochore intensity ( ± SD ) normalized to the values of GFP - Cyclin B2 - WT . Each dot represents one cell ( ≥ 30 cells from three independent experiments ) .

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(D) Representative immunofluorescence images of HeLa cells transfected with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for ACA (red) and DNA (blue). (E) Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2-WT/4A treated as in D. Bars represent the mean kinetochore intensity (±SD) normalized to the values of GFP-Cyclin B2-WT. Each dot represents one cell (≥30 cells from three independent experiments).


( A ) HeLa cells were transfected with siControl or siCyclin B2 . At 36 h after transfection , cells were collected and lysed . The samples were resolved by SDS - PAGE and analyzed by Western blotting using anti - Cyclin B2 antibody , anti - Cyclin B1 antibody , and anti - tubulin

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(A) HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were collected and lysed. The samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B2 antibody, anti-Cyclin B1 antibody, and anti-tubulin antibody


( B ) Representative stills illustrating mitotic progression in mCherry - H2B expressed cells treated with Control siRNA or Cyclin B2 siRNA . Images were acquired at the indicated time points after the start of NEBD . The arrow indicates lagging

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(B) Representative stills illustrating mitotic progression in mCherry-H2B expressed cells treated with Control siRNA or Cyclin B2 siRNA. Images were acquired at the indicated time points after the start of NEBD. The arrow indicates lagging chromosomes


( C ) Bar graph illustrating the percentage of cells with normal anaphase or with anaphase lagging chromosomes in cells treated as in B . Bars are means ± SD ( ≥ 30 cells from three independent experiments )

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(C) Bar graph illustrating the percentage of cells with normal anaphase or with anaphase lagging chromosomes in cells treated as in B. Bars are means ±SD (≥30 cells from three independent experiments)


( D ) Scatter graph of the time from NEBD to the beginning of anaphase in cells treated as in B ( n = 30 cells )

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(D) Scatter graph of the time from NEBD to the beginning of anaphase in cells treated as in B (n = 30 cells)


( E ) Representative stills illustrating mitotic progression in mCherry - H2B expressed cells depleted of endogenous Cyclin B2 and rescued with GFP - Cyclin B2 - WT or GFP - Cyclin B2 - 4A . Images were acquired at the indicated time points after the start of

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(E) Representative stills illustrating mitotic progression in mCherry-H2B expressed cells depleted of endogenous Cyclin B2 and rescued with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. Images were acquired at the indicated time points after the start of NEBD


( F ) Bar graph illustrating the percentage of cells with normal anaphase and cells with anaphase lagging chromosomes in cells treated as in E . Bars are means ± SD ( ≥ 30 cells from three independent experiments )

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(F) Bar graph illustrating the percentage of cells with normal anaphase and cells with anaphase lagging chromosomes in cells treated as in E. Bars are means ±SD (≥30 cells from three independent experiments)


Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2 . After 36 h of transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained for Mad2 ( green ) in D , ACA ( red ) , and DNA ( blue ) Representative immunofluorescence images of HeLa cells co - transfected with siCyclin B2 plus GFP - tagged Cyclin B2 - WT or Cyclin B2 - 4A . At 36 h post - transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained for Mad2 ( red ) in DNA ( blue )

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Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (green) in D, ACA (red), and DNA (blue) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (red) in DNA (blue)


Scatter graphs illustrating kinetochore intensity of Mad2 ( cells treated as in A and B Bars represent the mean kinetochore intensity ( ± SD ) normalized to the values of the siControl

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Scatter graphs illustrating kinetochore intensity of Mad2 ( cells treated as in A and B Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group


Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2 . After 36 h of transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained fo Cdc20 ( green ) in D , ACA ( red ) , and DNA ( blue ) E ) Representative immunofluorescence images of HeLa cells co - transfected with siCyclin B2 plus GFP - tagged Cyclin B2 - WT or Cyclin B2 - 4A . At 36 h post - transfection , cells were treated with nocodazole for 2 h . Then cells were fixed and co - stained fo Cdc20 ( red ) in E , and DNA ( blue )

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Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (green) in D, ACA (red), and DNA (blue) E) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (red) in E, and DNA (blue)


Scatter graphs illustrating kinetochore intensity o Cdc20 ( F , cells treated as in D and E ) Bars represent the mean kinetochore intensity ( ± SD ) normalized to the values of the siControl

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Scatter graphs illustrating kinetochore intensity o Cdc20 (F, cells treated as in D and E) Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group


( G ) Bar graph illustrating the mitotic index in cells treated with low concentrations of nocodazole for 12

(G) Bar graph illustrating the mitotic index in cells treated with low concentrations of nocodazole for 12 h


( H ) Immunoprecipitation assay performed with Cdc20 antibody . HeLa cells were transfected with siControl or siCyclin B2 . At 36 h after transfection , cells were arrested in mitosis , collected , and lysed . Cell lysates were incubated with Protein A / G beads bound with Cdc20 antibody . Following extensive washes , immunoprecipitates were resolved by SAS - PAGE and analyzed by Western blotting using anti - Cdc20 , anti - BubR1 , anti - Bub3 , and anti - Mad2

(H) Immunoprecipitation assay performed with Cdc20 antibody. HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were arrested in mitosis, collected, and lysed. Cell lysates were incubated with Protein A/G beads bound with Cdc20 antibody. Following extensive washes, immunoprecipitates were resolved by SAS-PAGE and analyzed by Western blotting using anti-Cdc20, anti-BubR1, anti-Bub3, and anti-Mad2 antibodies


Subcellular localization of NeoR and NucNeoR and T cell recognition of NeoR ‐ and NucNeoR ‐ expressing cells . RCC1 . 24 cells were transfected with the NeoR ( A ) and NucNeoR ( B ) expression constructs . After 2 days , the subcellular localization of the proteins was analyzed by immunofluorescence using the anti c ‐ myc antibody 9E10 . While NeoR was evenly distributed in nucleus and cytoplasm ( a ) , NucNeoR was confined to the cell nucleus ( b ) . Daylight photographs of the stained cells are shown in ( a ' ) and ( b ' ) .

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Subcellular localization of NeoR and NucNeoR and T cell recognition of NeoR‐ and NucNeoR‐expressing cells. RCC1.24 cells were transfected with the NeoR (A) and NucNeoR (B) expression constructs. After 2 days, the subcellular localization of the proteins was analyzed by immunofluorescence using the anti c‐myc antibody 9E10. While NeoR was evenly distributed in nucleus and cytoplasm (a), NucNeoR was confined to the cell nucleus (b). Daylight photographs of the stained cells are shown in (a') and (b').


( C ) NeoR ‐ and NucNeoR ‐ transfected RCC1 . 24 cells were treated with 100 U / mL IFN ‐ γ for 48 h to induce MHC class II expression . Subsequently , the cells were washed and co ‐ cultured with the CD4 clone 20 ‐ 4 / A4 , which recognizes a peptide derived from NeoR on HLA ‐ DP3 . GM ‐ CSF secretion by the T cells was measured 20 h later by ELISA . Both transfectants were recognized after IFN ‐ γ treatment , indicating that directing NeoR into the cell nucleus does not abrogate antigen presentation .

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(C) NeoR‐ and NucNeoR‐transfected RCC1.24 cells were treated with 100 U/mL IFN‐γ for 48 h to induce MHC class II expression. Subsequently, the cells were washed and co‐cultured with the CD4 clone 20‐4/A4, which recognizes a peptide derived from NeoR on HLA‐DP3. GM‐CSF secretion by the T cells was measured 20 h later by ELISA. Both transfectants were recognized after IFN‐γ treatment, indicating that directing NeoR into the cell nucleus does not abrogate antigen presentation.


( D ) LCL1 . 11 cells were transfected with NeoR or NucNeoR expression plasmids and antigen presentation was assessed 48 h later with the NeoR ‐ specific T cell clone 20 ‐ 4 / A4 . Antigen ‐ transfected LCL1 . 11 cells were recognized irrespectively of NeoR localization , while untransfected cells or cells transfected with a GFP ‐ expressing control plasmid were not recognized .

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(D) LCL1.11 cells were transfected with NeoR or NucNeoR expression plasmids and antigen presentation was assessed 48 h later with the NeoR‐specific T cell clone 20‐4/A4. Antigen‐transfected LCL1.11 cells were recognized irrespectively of NeoR localization, while untransfected cells or cells transfected with a GFP‐expressing control plasmid were not recognized.


( A ) RCC1 . 24 ‐ NeoR and ‐ NucNeoR cells were incubated for 48 h with IFN ‐ γ and in addition with 7 . 5 mM 3 ‐ MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h . Subsequently , the cells were fixed with 0 . 5 % paraformaldehyde and tested with 20 ‐ 4 / A4 cells in a GM ‐ CSF release assay . Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing .

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(A) RCC1.24‐NeoR and ‐NucNeoR cells were incubated for 48 h with IFN‐γ and in addition with 7.5 mM 3‐MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h. Subsequently, the cells were fixed with 0.5% paraformaldehyde and tested with 20‐4/A4 cells in a GM‐CSF release assay. Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing.


( B ) LCL1 . 11 cells transfected with NeoR or NucNeoR expression plasmids were tested likewise . Presentation of endogenous NeoR and NucNeoR was blocked by all inhibitors , indicating that NeoR and NucNeoR presentation is dependent on autophagy and lysosomal processing .

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(B) LCL1.11 cells transfected with NeoR or NucNeoR expression plasmids were tested likewise. Presentation of endogenous NeoR and NucNeoR was blocked by all inhibitors, indicating that NeoR and NucNeoR presentation is dependent on autophagy and lysosomal processing.


( C ) To exclude toxic side effects of the inhibitors , LCL1 . 11 cells were incubated for 20 h with 200 ng / mL recombinant NeoR protein in the presence or absence of the inhibitors . Afterwards , residual protein and inhibitors were removed by washing and the cells were co ‐ cultured with the NeoR ‐ specific CD4 + T cell clone 20 ‐ 4 / A4 . The presentation of exogenous NeoR on MHC class II was not affected by 3 ‐ MA , but was impaired when lysosomal processing was blocked with chloroquine or leupeptin .

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(C) To exclude toxic side effects of the inhibitors, LCL1.11 cells were incubated for 20 h with 200 ng/mL recombinant NeoR protein in the presence or absence of the inhibitors. Afterwards, residual protein and inhibitors were removed by washing and the cells were co‐cultured with the NeoR‐specific CD4+ T cell clone 20‐4/A4. The presentation of exogenous NeoR on MHC class II was not affected by 3‐MA, but was impaired when lysosomal processing was blocked with chloroquine or leupeptin.


( D ) LCL1 . 11 cells were transfected with siRNA specific for Atg12 or GFP , and Atg12 mRNA levels were determined 24 h later by qPCR . The relative amount of Atg12 mRNA was reduced approximately fourfold .

(D) LCL1.11 cells were transfected with siRNA specific for Atg12 or GFP, and Atg12 mRNA levels were determined 24 h later by qPCR. The relative amount of Atg12 mRNA was reduced approximately fourfold.


( E ) At 24 h after siRNA transfection , NeoR and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later . In cells transfected with Atg12 siRNA , but not in mock ‐ transfected cells or cells transfected with GFP siRNA , antigen presentation of both NeoR and NucNeoR was greatly reduced .

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(E) At 24 h after siRNA transfection, NeoR and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later. In cells transfected with Atg12 siRNA, but not in mock‐transfected cells or cells transfected with GFP siRNA, antigen presentation of both NeoR and NucNeoR was greatly reduced.


( A ) RCC1 . 24 cells were transfected with the NES ‐ GFP ‐ NeoR ‐ GFP ‐ NLS expression construct and subcellular localization of the fusion protein was monitored by UV fluorescence . As compared to untreated cells ( a ) , cells treated with 8 nM LMB for 20 h ( b ) showed a strong accumulation of the protein in the nucleus . ( a ' ) and ( b ' ) are the corresponding daylight photographs of the transfected cells .

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(A) RCC1.24 cells were transfected with the NES‐GFP‐NeoR‐GFP‐NLS expression construct and subcellular localization of the fusion protein was monitored by UV fluorescence. As compared to untreated cells (a), cells treated with 8 nM LMB for 20 h (b) showed a strong accumulation of the protein in the nucleus. (a') and (b') are the corresponding daylight photographs of the transfected cells.


( B ) NeoR ‐ and NucNeoR ‐ transfected LCL1 . 11 cells were either left untreated or incubated for 20 h with 8 nM LMB . Subsequently , the cells were fixed with 0 . 5 % paraformaldehyde and probed with the NeoR ‐ specific T cells . LMB treatment had no significant effect on the presentation of NeoR or NucNeoR on MHC class II , indicating that CRM1 ‐ dependent nuclear export is not involved in the presentation of this nuclear antigen on MHC class II .

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(B) NeoR‐ and NucNeoR‐transfected LCL1.11 cells were either left untreated or incubated for 20 h with 8 nM LMB. Subsequently, the cells were fixed with 0.5% paraformaldehyde and probed with the NeoR‐specific T cells. LMB treatment had no significant effect on the presentation of NeoR or NucNeoR on MHC class II, indicating that CRM1‐dependent nuclear export is not involved in the presentation of this nuclear antigen on MHC class II.


( G ) Immunostaining of pS6 ( Ser240 / 244 ) in skin lesions from control and LL37 - induced mice . Bottom right panels , magnified images of boxed areas . DAPI staining ( blue ) indicates nuclear localization . Scale bar : 50 μm . Right panel , the quantification of relative fluorescence intensity for pS6 ( Ser240 / 244 ) in control and LL37 group ( n = 5 for each group ) . Data represent the mean ± SEM . * * P < 0 . 01 . two - tailed unpaired Student ' s t - test was used .

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(G) Immunostaining of pS6 (Ser240/244) in skin lesions from control and LL37-induced mice. Bottom right panels, magnified images of boxed areas. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. Right panel, the quantification of relative fluorescence intensity for pS6 (Ser240/244) in control and LL37 group (n=5 for each group). Data represent the mean ± SEM. **P < 0.01. two-tailed unpaired Student's t-test was used.


( D ) HE staining of lesional skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle ( n = 6 for each group ) . Scale bar : 50 μm . ( E ) Dermal infiltrating cells were quantified ( n = 6 for each group ) for each high power field ( HPF ) .

(D) HE staining of lesional skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle (n=6 for each group). Scale bar: 50 μm. (E) Dermal infiltrating cells were quantified (n = 6 for each group) for each high power field (HPF).


( F ) The mRNA expression levels of Tnf - α , Il6 , Mmp9 and Vegf in skin lesions ( n = 6 for each group ) .

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(F) The mRNA expression levels of Tnf-α, Il6, Mmp9 and Vegf in skin lesions (n = 6 for each group).


( I ) HE staining of lesional skin sections from LL37 or control mice topically applied with RAPA or vehicle . Scale bar : 50 μm . ( J ) Dermal infiltrating cells were quantified ( n = 6 for each group ) . Data represent the mean ± SEM . * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test was used .

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(I) HE staining of lesional skin sections from LL37 or control mice topically applied with RAPA or vehicle. Scale bar: 50 μm. (J) Dermal infiltrating cells were quantified (n = 6 for each group). Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.


( A ) The back skins of WT ( Tsc2 + / + ) and Tsc2 + / - mice were intradermally injected with LL37 or control vehicle ( n = 5 for each group ) . Pictures were taken on day 0 . 5 , 2 , 5 , 8 , 12 after the first LL37 injection . The mouse experiments were repeated for three times , and 5 mice were included in each group for each time . The results of a representative mouse experiment were displayed . Scale bar : 2 mm .

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(A) The back skins of WT (Tsc2+/+) and Tsc2+/- mice were intradermally injected with LL37 or control vehicle (n=5 for each group). Pictures were taken on day 0.5, 2, 5, 8, 12 after the first LL37 injection. The mouse experiments were repeated for three times, and 5 mice were included in each group for each time. The results of a representative mouse experiment were displayed. Scale bar: 2 mm.


( A ) The mRNA expression level of human cathelicidin ( CAMP ) in skin lesions from healthy individuals ( n = 10 ) and patients with rosacea ( n = 15 ) .

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(A) The mRNA expression level of human cathelicidin (CAMP) in skin lesions from healthy individuals (n = 10) and patients with rosacea (n = 15). Data


( G ) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 ( 4 μM ) ± RAPA for 24 h . DAPI staining ( blue ) indicates nuclear localization . Scale bar : 50 μm . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM .

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(G) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM.


( E ) Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC - labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS washed , then live cells were analyzed by fluorescent microscope . Scale bar : 50 μm .

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(E) Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC-labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS washed, then live cells were analyzed by fluorescent microscope. Scale bar: 50 μm.


( F , G ) Primary human keratinocytes were incubated with sLL37 - flag or LL37 - flag for 1 h . Cell lysates were immunoprecipitated with anti - flag or anti - TLR2 antibodies , showing an interaction between LL37 and TLR2 . Medium , the medium of primary human keratinocytes treated with sLL37 - flag or LL37 - flag . All results are representative of at least 3 independent experiments .

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(F, G) Primary human keratinocytes were incubated with sLL37-flag or LL37-flag for 1 h. Cell lysates were immunoprecipitated with anti-flag or anti-TLR2 antibodies, showing an interaction between LL37 and TLR2. Medium, the medium of primary human keratinocytes treated with sLL37-flag or LL37-flag. All results are representative of at least 3 independent experiments.


( E ) Immunostaining of phospho - p65 ( p - p65 ) in skin sections . Bottom right panels , magnified images of dotted line areas . Epi , epidermis . Der , dermis . Scale bar : 50 μm . ( F ) Quantification of relative fluorescence intensity for p - p65 in epidermis ( n = 6 ) .

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(E) Immunostaining of phospho-p65 (p-p65) in skin sections. Bottom right panels, magnified images of dotted line areas. Epi, epidermis. Der, dermis. Scale bar: 50 μm. (F) Quantification of relative fluorescence intensity for p-p65 in epidermis (n=6).


( G ) The mRNA expression levels of NF - κB family of transcription factors ( Nfκb1 , Nfκb1 , Rela and Relb ) in mice skin lesions ( n = 6 for each group ) .

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(G) The mRNA expression levels of NF-κB family of transcription factors (Nfκb1, Nfκb1, Rela and Relb) in mice skin lesions (n=6 for each group).


( I ) IHC of p65 on skin sections from HS and rosacea . Scale bar , 50 μm . ( J ) Percentage of nuclear p65 positive cells in the epidermis from HS ( n = 8 ) and rosacea ( n = 13 ) . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM . * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test ( F and G ) or two - tailed unpaired Student ' s t - test ( J ) was used .

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(I) IHC of p65 on skin sections from HS and rosacea. Scale bar, 50 μm. (J) Percentage of nuclear p65 positive cells in the epidermis from HS (n = 8) and rosacea (n = 13). All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test (F and G) or two-tailed unpaired Student's t-test (J) was used.


( B ) The mRNA expression levels of mouse chemokines ( Cxcl11 , Cxcl12 , Ccl2 and Ccl3 ) in skin lesions ( n = 6 for each group ) .

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(B) The mRNA expression levels of mouse chemokines (Cxcl11, Cxcl12, Ccl2 and Ccl3) in skin lesions (n=6 for each group).


( E ) Heatmap of upregulated chemokines and cytokines in rosacea skin lesions determined by RNA - sequencing . All results are representative of at least 3 independent experiments . Data represent the mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 . 1 - way ANOVA with Bonferroni ' s post hoc test was used .

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(E) Heatmap of upregulated chemokines and cytokines in rosacea skin lesions determined by RNA-sequencing. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. *P < 0.05, **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.


Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients . ( C ) The change of patients ' CEA score showed in scatter histogram at 0 week and 4th week . ( D ) The change of patients ' IGA score showed in scatter histogram at 0 week and 4th week . " The Change value < 0 " indicates that skin lesions show positive improvement after treatment ; " The Change value > 0 " indicates skin lesions show exacerbation after treatment ; " The Change value = 0 " indicates that skin lesions show no change after treatment ; Data represent the mean ± SEM , and two - tailed unpaired Student ' s t - test was used ( C , D ) .

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Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients. (C) The change of patients' CEA score showed in scatter histogram at 0 week and 4th week. (D) The change of patients' IGA score showed in scatter histogram at 0 week and 4th week. "The Change value < 0" indicates that skin lesions show positive improvement after treatment; "The Change value > 0" indicates skin lesions show exacerbation after treatment; "The Change value = 0" indicates that skin lesions show no change after treatment; Data represent the mean ± SEM, and two-tailed unpaired Student's t-test was used (C, D).


( A ) ARPE - 19 cells were incubated in the presence of DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) , or starved ( EBSS ) for 16 h . Cells were fixed , permeabilized and stained with an antibody against TFE3 . Scale bar , 10 µm . Data are representative of three independent experiments .

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(A) ARPE-19 cells were incubated in the presence of DMSO (Control), Tunicamycin (5 µg/ml), or starved (EBSS) for 16 h. Cells were fixed, permeabilized and stained with an antibody against TFE3. Scale bar, 10 µm. Data are representative of three independent experiments.


( B ) Immunoblot of protein lysates of ARPE - 19 cells treated as shown in ( A ) . Data are representative of three independent experiments .

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(B) Immunoblot of protein lysates of ARPE-19 cells treated as shown in (A). Data are representative of three independent experiments.


( C ) ARPE - 19 cells incubated with Tunicamycin as indicated in ( A ) were immunostained with antibody against CHOP . Scale bar , 10 µm . Data are representative of three independent experiments .

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(C) ARPE-19 cells incubated with Tunicamycin as indicated in (A) were immunostained with antibody against CHOP. Scale bar, 10 µm. Data are representative of three independent experiments.


( D ) Quantification of the percentage of ARPE - 19 cells with nuclear TFE3 upon Tunicamycin ( Tun . ) or starvation ( Strv . ) treatments as indicated in ( A ) ( mean ± SD of three independent experiments , one - way ANOVA analysis versus Control treated cells , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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(D) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or starvation (Strv.) treatments as indicated in (A) (mean ± SD of three independent experiments, one-way ANOVA analysis versus Control treated cells, ***p < 0.001; n>400 cells per condition).


( E ) Wild - type mouse embryonic fibroblast ( MEF ) cells were incubated in the presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) for the indicated times . Cells were fixed , permeabilized and stained with antibodies against TFE3 ( green ) and CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments .

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

(E) Wild-type mouse embryonic fibroblast (MEF) cells were incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.


( F ) Wild - type MEF cells were incubated in the presence of DMSO ( Control ) or BFA ( 2 . 5 µg / ml ) for 16 h . Cells were fixed , permeabilized and stained with antibodies against TFE3 ( green ) and CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments . ( G ) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO ( Control ) or BFA 16 h treatment ( mean ± SD of two independent experiments , student ' s t - test analysis versus DMSO treated cells , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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 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

(F) Wild-type MEF cells were incubated in the presence of DMSO (Control) or BFA (2.5 µg/ml) for 16 h. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(G) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or BFA 16 h treatment (mean ± SD of two independent experiments, student's t-test analysis versus DMSO treated cells, ***p < 0.001; n>400 cells per condition).


( H ) Immunoblots of the subcellular distribution of TFE3 and TFEB in MEF cells incubated with DMSO ( Ctrl . ) or Tunicamycin ( Tun . ) for 16 h using TFE3 , TFEB , Lamp1 ( as a lysosomal membrane marker ) , and H3b ( as a nuclear marker ) . Data are representative of three independent experiments .

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(H) Immunoblots of the subcellular distribution of TFE3 and TFEB in MEF cells incubated with DMSO (Ctrl.) or Tunicamycin (Tun.) for 16 h using TFE3, TFEB, Lamp1 (as a lysosomal membrane marker), and H3b (as a nuclear marker). Data are representative of three independent experiments.


( A ) Immunoblot of protein lysates from wild - type , PERK - KO and GCN2 - KO MEF cells incubated in the presence of DMSO ( Control ) , Tunicamycin ( 0 . 1 µg / ml ) , or starved in EBSS for 16 h . Data are representative of three independent experiments .

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(A) Immunoblot of protein lysates from wild-type, PERK-KO and GCN2-KO MEF cells incubated in the presence of DMSO (Control), Tunicamycin (0.1 µg/ml), or starved in EBSS for 16 h. Data are representative of three independent experiments.


( B - D ) Wild - type ( B ) , GCN2 - KO ( C ) , and PERK - KO ( D ) MEF cells incubated in the presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) as indicated in ( A ) . Cells were fixed , permeabilized and stained with antibodies against TFE3 ( green ) and CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments . ( E - F ) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO ( Control ) , Tunicamycin treatments for 16 h ( E ) , or starvation for 2 h ( F ) ( mean ± SD of three independent experiments , one - way ANOVA analysis versus DMSO treated cells , ns = not significant , * * * p < 0 . 001 ; n > 400 cells per 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 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 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

(B-D) Wild-type (B), GCN2-KO (C), and PERK-KO (D) MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) as indicated in (A). Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(E-F) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control), Tunicamycin treatments for 16 h (E), or starvation for 2 h (F) (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated cells, ns= not significant, ***p < 0.001; n>400 cells per condition).


( G ) Quantification of phospho - 4EBP1 protein levels in wild - type and GCN2 - KO MEF cells upon starvation for 2 h ( the mean ± SD of the fold increase of the phospho - 4EBP1 to total 4EBP1 ratio from three independent experiments , one - way ANOVA analysis versus starvation treated wild - type cells , * * * 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

(G) Quantification of phospho-4EBP1 protein levels in wild-type and GCN2-KO MEF cells upon starvation for 2 h (the mean ± SD of the fold increase of the phospho-4EBP1 to total 4EBP1 ratio from three independent experiments, one-way ANOVA analysis versus starvation treated wild-type cells, ***p < 0.001).


( H ) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO ( Control ) or Torin - 1 ( 250 nM ) treatments for 1 h ( mean ± SD of three independent experiments , one - way ANOVA analysis versus DMSO treated wild - type cells , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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

(H) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or Torin-1 (250 nM) treatments for 1 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated wild-type cells, ***p < 0.001; n>400 cells per condition).


( A ) ARPE - 19 cells infected with either adenovirus expressing TFE3 - WT - Myc or TFE3 - S321A - Myc for 16h . Cells were fixed , permeabilized and stained with antibodies against Myc . Scale bar , 10 µm . Quantification of the percentage of ARPE - 19 cells with nuclear TFE3 - Myc ( means ± SD of 3 independent experiments , students t - test , * * * * p < 0 . 0001 ; n > 400 cells per 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ARPE-19 cells infected with either adenovirus expressing TFE3-WT-Myc or TFE3-S321A-Myc for 16h. Cells were fixed, permeabilized and stained with antibodies against Myc. Scale bar, 10 µm. Quantification of the percentage of ARPE-19 cells with nuclear TFE3-Myc (means ± SD of 3 independent experiments, students t-test, ****p < 0.0001; n>400 cells per condition).


( B ) Immunoblots of protein lysates from ARPE - 19 cells infected as indicated in ( A ) . Data are representative of three independent experiments .

(B) Immunoblots of protein lysates from ARPE-19 cells infected as indicated in (A). Data are representative of three independent experiments.


( C ) Immunoblots of TFE3 - Ser321 phosphorylation state in nuclear fractions of ARPE - 19 cells incubated with DMSO ( Ctrl . ) , Tunicamycin ( Tun . ) , or Torin - 1 . Data are representative of three independent experiments .

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(C) Immunoblots of TFE3-Ser321 phosphorylation state in nuclear fractions of ARPE-19 cells incubated with DMSO (Ctrl.), Tunicamycin (Tun.), or Torin-1. Data are representative of three independent experiments.


( D ) Immunoblots of protein of a GST pull - down from nuclear fractions of MEF cells treated with DMSO or Tunicamycin ( 5 µg / ml ) ( Tun . ) for 16 h . Data are representative of three independent experiments .

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(D) Immunoblots of protein of a GST pull-down from nuclear fractions of MEF cells treated with DMSO or Tunicamycin (5 µg/ml) (Tun.) for 16 h. Data are representative of three independent experiments.


( E ) Immunoblots of protein lysates from ARPE - 19 cells incubated in the presence of DMSO ( Ctrl . ) , Tunicamycin ( 5 µg / ml ) ( Tun . ) , or starved in EBSS ( Strv . ) for 16 h . Data are representative of three independent experiments .

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

(E) Immunoblots of protein lysates from ARPE-19 cells incubated in the presence of DMSO (Ctrl.), Tunicamycin (5 µg/ml) (Tun.), or starved in EBSS (Strv.) for 16 h. Data are representative of three independent experiments.


( F ) Quantification of the percentage of ARPE - 19 cells with nuclear TFE3 upon Tunicamycin ( Tun . ) or Tunicamycin + FK506 treatments for 16 h ( mean ± SD of three independent experiments , one - way ANOVA analysis versus Tunicamycin treated cells , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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

(F) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Tunicamycin + FK506 treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus Tunicamycin treated cells, ***p < 0.001; n>400 cells per condition).


( G ) Quantification of the percentage of calcineurin depleted ARPE - 19 cells with nuclear TFE3 upon Tunicamycin ( Tun . ) or Starvation ( Strv . ) treatments for 16 h ( mean ± SD of three independent experiments , one - way ANOVA analysis versus siNon - target treated cells within the indicated treatment , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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(G) Quantification of the percentage of calcineurin depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Starvation (Strv.) treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment, ns= not significant, *p < 0.05, **p < 0.01, ***p < 0.001; n>400 cells per condition).


( H ) Immunoblots of protein lysates from ARPE - 19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB . Data are representative of three independent experiments .

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(H) Immunoblots of protein lysates from ARPE-19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB. Data are representative of three independent experiments.


( I ) Relative Quantitative Real - Time PCR analysis of PPP3CA and PPP3CB mRNA transcript levels in ARPE - 19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analsysis versus the siNon - Target ( siNT ) treated cells , * * * 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

(I) Relative Quantitative Real-Time PCR analysis of PPP3CA and PPP3CB mRNA transcript levels in ARPE-19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analsysis versus the siNon-Target (siNT) treated cells, ***p < 0.001).


( J ) Quantification of the percentage of ATF4 depleted ARPE - 19 cells with nuclear TFE3 upon Tunicamycin treatment for 16 h ( mean ± SD of two independent experiments , one - way ANOVA analysis versus siNon - target treated cells within the indicated treatment * * * p < 0 . 001 ; n > 400 cells per condition ) .

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(J) Quantification of the percentage of ATF4 depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin treatment for 16 h (mean ± SD of two independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment***p < 0.001; n>400 cells per condition).


( A ) Relative Quantitative Real - Time PCR analysis of TFEB and TFE3 mRNA transcript levels in ARPE - 19 cells depleted of TFEB and TFE3 ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , student ' s t - test versus siNon - Target ( siNT ) treated cells , * * * 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

(A) Relative Quantitative Real-Time PCR analysis of TFEB and TFE3 mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, student's t-test versus siNon-Target (siNT) treated cells, ***p < 0.001).


( B ) Relative Quantitative Real - Time PCR analysis of MCOLN1 , ATP6V1C1 , HEXA and UVRAG mRNA transcript levels in ARPE - 19 cells depleted of TFEB and TFE3 upon incubation with DMSO ( Control ) or Tunicamycin ( 5 µg / ml ) for 16 h ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analysis versus the same treatment condition in the siNon - Target ( siNT ) treated cells or versus the DMSO condition in siNon - Target ( siNT ) treated cells , * 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 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

(B) Relative Quantitative Real-Time PCR analysis of MCOLN1, ATP6V1C1, HEXA and UVRAG mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 upon incubation with DMSO (Control) or Tunicamycin (5 µg/ml) for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the same treatment condition in the siNon-Target (siNT) treated cells or versus the DMSO condition in siNon-Target (siNT) treated cells, *p < 0.05, **p < 0.01, ***p < 0.001).


( A ) Immunoblots of protein lysates from ARPE - 19 cells infected with either adenovirus expressing Null , TFEB - S211A , or TFE3 upon incubation with DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) or starved in EBSS for 16 h . Data are representative of five independent experiments . ( B ) Quantification of ATF4 , CHOP and GADD34 protein levels in ARPE - 19 cells treated as indicated in ( A ) ( mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments , one - way ANOVA analysis versus the corresponding treatment condition in the adenovirus - Null infected cells , * 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 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

(A) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A, or TFE3 upon incubation with DMSO (Control), Tunicamycin (5 µg/ml) or starved in EBSS for 16 h. Data are representative of five independent experiments.(B) Quantification of ATF4, CHOP and GADD34 protein levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, *p < 0.05, **p < 0.01, ***p < 0.001).


( C ) Relative Quantitative Real - Time PCR analysis of ATF4 , CHOP , GADD34 and MCOLN1 mRNA transcript levels in ARPE - 19 cells treated as indicated in ( A ) ( mean ± SD of the RNA fold change of indicated genes normalized to actin mRNA from three independent experiments , one - way ANOVA analysis versus the corresponding treatment condition in the adenovirus - Null infected cells , * * p < 0 . 01 , * * * p < 0 . 001 ) .

(C) Relative Quantitative Real-Time PCR analysis of ATF4, CHOP, GADD34 and MCOLN1 mRNA transcript levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the RNA fold change of indicated genes normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, **p < 0.01, ***p < 0.001).


( A - D ) Relative Quantitative Real - Time PCR analysis of ATF4 ( A ) , ASNS ( B ) , CAT1 ( C ) and xCT ( D ) mRNA transcript levels in ATF4 - depleted ARPE - 19 cells infected with either adenovirus expressing Null or TFEB - S211A upon incubation with DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) , or starved in EBSS for 16 h ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analysis versus the corresponding treatment condition in the adenovirus - Null infected and siRNA Non - Target ( siNT ) treated cells , * 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 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

(A-D) Relative Quantitative Real-Time PCR analysis of ATF4 (A), ASNS (B), CAT1 (C) and xCT (D) mRNA transcript levels in ATF4-depleted ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A upon incubation with DMSO (Control), Tunicamycin (5 µg/ml), or starved in EBSS for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected and siRNA Non-Target (siNT) treated cells, *p < 0.05, ***p < 0.001).