F ) Co - immunoprecipitation analysis to elucidate the interaction between Setdb1 and HP1 . Whole - cell extracts of HEK293T cells expressing 3xFlag - Setdb1 or 3xFlag - Setdb1 mutants were subjected to immunoprecipitation with the anti - HP1β antibody The immunocomplex was analyzed by immunoblotting with the indicated antibodies .

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F) Co-immunoprecipitation analysis to elucidate the interaction between Setdb1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Setdb1 or 3xFlag-Setdb1 mutants were subjected to immunoprecipitation with the anti-HP1β antibody The immunocomplex was analyzed by immunoblotting with the indicated antibodies.


G ) Protein decay analysis of HP1 interaction - defective Setdb1 mutant . mESC lines stably expressing 3xFlag - Setdb1 or 3xFlag - Setdb1 - 3A were treated with CHX and MG132 for the indicated times . Stacked bar plots show the summarized protein amounts of Setdb1 ( right ) . Data represented as mean ± SD ( n = 4 , biological replicates ) . * * p < 0 . 01 ( Tukey ' s honestly significant difference test ) .

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G) Protein decay analysis of HP1 interaction-defective Setdb1 mutant. mESC lines stably expressing 3xFlag-Setdb1 or 3xFlag-Setdb1-3A were treated with CHX and MG132 for the indicated times. Stacked bar plots show the summarized protein amounts of Setdb1 (right). Data represented as mean ± SD (n = 4, biological replicates). **p < 0.01 (Tukey's honestly significant difference test).


B ) Protein expression levels of H3K9 MTs and histone modification levels in HP1 - cTKO cells were monitored by immunoblotting . The intensities were quantified using ImageJ software and summarized on the right . The thin gray line and bold colored line show individual data and means of the experimental replicates ( n ≥ 3 , biological replicates ) , respectively .

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B) Protein expression levels of H3K9 MTs and histone modification levels in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and means of the experimental replicates (n ≥ 3, biological replicates), respectively.


C ) Comparison of H3K9me3 levels between HP1 - deficient cells and Suv39h / Setdb1 - deficient cells . H3K9me levels were quantified using ImageJ software and are summarized at the bottom ; the horizontal line represents the mean intensity of each cell . More than 75 nuclei were counted for each staining . * * p < 0 . 01 ( Tukey ' s honestly significant difference test ) .

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C) Comparison of H3K9me3 levels between HP1-deficient cells and Suv39h/Setdb1-deficient cells. H3K9me levels were quantified using ImageJ software and are summarized at the bottom; the horizontal line represents the mean intensity of each cell. More than 75 nuclei were counted for each staining. **p < 0.01 (Tukey's honestly significant difference test).


D ) Electron microscopy images of HP1 - cTKO cells in the absence of 4OHT ( left ) and of cells treated with 4OHT for 4 days ( right ) . Boxes represent enlarged images .

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D) Electron microscopy images of HP1-cTKO cells in the absence of 4OHT (left) and of cells treated with 4OHT for 4 days (right). Boxes represent enlarged images.


B ) Subcellular distribution of H3K9 MTs in the HP1 - cTKO cells was examined by immunoblotting . α - Tubulin , Ezh2 , and histone H3 were used as representative markers for each fraction . Stacked bar plots show the abundance ratios of H3K9 MTs among the three fractions ( right ) .

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B) Subcellular distribution of H3K9 MTs in the HP1-cTKO cells was examined by immunoblotting. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction. Stacked bar plots show the abundance ratios of H3K9 MTs among the three fractions (right).


B , Elucidation of protein - protein interaction between HP1 and H3K9 MTs by co - immunoprecipitation analysis . Whole - cell extracts of HEK293T cells co - expressing HA - HP1β mutants and epitope - tagged proteins of interest were subjected to immunoprecipitation with the anti - HA tag antibody . The immunocomplex was analyzed by immunoblotting with the indicated antibodies . The intensities were quantified using ImageJ software , normalized , and shown on the top .

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B, Elucidation of protein-protein interaction between HP1 and H3K9 MTs by co-immunoprecipitation analysis. Whole-cell extracts of HEK293T cells co-expressing HA-HP1β mutants and epitope-tagged proteins of interest were subjected to immunoprecipitation with the anti-HA tag antibody. The immunocomplex was analyzed by immunoblotting with the indicated antibodies. The intensities were quantified using ImageJ software, normalized, and shown on the top.


E ) Evaluation of HP1β mutants for maintaining H3K9me2 / 3 and ensuring protein stability of H3K9 MTs . H3K9me2 / 3 levels and protein levels of H3K9 MTs in HP1 - cTKO lines expressing 3xFlag - hHP1β mutants were compared before and after 4OHT treatment . The intensities were quantified with ImageJ software and are summarized on the right . Data represented as mean ± SD ( n > 3 , biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 ( Welch ' s test ) .

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

E) Evaluation of HP1β mutants for maintaining H3K9me2/3 and ensuring protein stability of H3K9 MTs. H3K9me2/3 levels and protein levels of H3K9 MTs in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).


B ) Protein expression levels of Jmjd1a / b in HP1 - cTKO cells were monitored by immunoblotting . The intensities were quantified using ImageJ software and summarized on the right . The thin gray line and bold colored line show individual data and the mean of experimental replicates , respectively .

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B) Protein expression levels of Jmjd1a/b in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and the mean of experimental replicates, respectively.


C ) Evaluation of HP1β mutants for ensuring protein stability of Jmjd1a / b . Protein levels of Jmjd1a / b in HP1 - cTKO lines expressing 3xFlag - hHP1β mutants were compared before and after 4OHT treatment . The intensities were quantified with ImageJ software and are summarized on the right . Data represented as mean ± SD ( n > 3 , biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 ( Welch ' s test ) .

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

C) Evaluation of HP1β mutants for ensuring protein stability of Jmjd1a/b. Protein levels of Jmjd1a/b in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).


C ) Impact of Suv39h / Setdb1 / G9a depletion on HP1 - mediated protein stability regulation of Jmjd1a / b . Jmjd1a / b were destabilized in mESCs lacking Suv39h / Setdb1 / G9a , even though HP1 was present .

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C) Impact of Suv39h/Setdb1/G9a depletion on HP1-mediated protein stability regulation of Jmjd1a/b. Jmjd1a/b were destabilized in mESCs lacking Suv39h/Setdb1/G9a, even though HP1 was present.


D ) Functional evaluation of H3K9me2 / 3 for confining HP1 to chromatin . Subcellular localizations of HP1α , β and γ in TT2 and mESCs lacking Suv39h / Setdb1 / G9a were compared . α - Tubulin , Ezh2 , and histone H3 were used as representative markers for each fraction .

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D) Functional evaluation of H3K9me2/3 for confining HP1 to chromatin. Subcellular localizations of HP1α, β and γ in TT2 and mESCs lacking Suv39h/Setdb1/G9a were compared. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction.


Growth suppression in cancer cells after 72h of treatment with APR - 246 + / - MRP1 inhibitor MK - 571 as shown by the WST - 1 assay ( n ≥ 3 , n shown in Table S1 ) . Cross in heatmap indicates no data available .

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Growth suppression in cancer cells after 72h of treatment with APR-246 +/- MRP1 inhibitor MK-571 as shown by the WST-1 assay (n ≥ 3, n shown in Table S1). Cross in heatmap indicates no data available.


Growth suppression in human dermal fibroblasts ( HDF ) by WST - 1 assay after 72h of with APR - 246 + / - MK - 571 ( n = 3 ) .

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Growth suppression in human dermal fibroblasts (HDF) by WST-1 assay after 72h of with APR-246 +/- MK-571 (n = 3).


Mean IC50 values ( µM ) for APR - 246 + / - 20 μM MK - 571 of 21 cell lines . * p < 0 . 0001 , Wilcoxon test . Each dot represents one cell line . Mean IC50 values and n shown in Table S1 . ZIP synergy scores of most synergistic area sub - grouped according to TP53 gene status for 21 cell lines . Each dot represents one cell line . Scores and n are shown in Table S1 .

O O 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 O O O O O O O O O O O O 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 values (µM) for APR-246 +/- 20 μM MK-571 of 21 cell lines. *p < 0.0001, Wilcoxon test. Each dot represents one cell line. Mean IC50 values and n shown in Table S1. ZIP synergy scores of most synergistic area sub-grouped according to TP53 gene status for 21 cell lines. Each dot represents one cell line. Scores and n are shown in Table S1.


Sub - G1 DNA content of OVCAR - 3 cells ( R248Q ) after APR - 246 + / - MK - 571 treatment as assessed by propidium iodide staining at 48h ( n = 3 ) .

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Sub-G1 DNA content of OVCAR-3 cells (R248Q) after APR-246 +/- MK-571 treatment as assessed by propidium iodide staining at 48h (n = 3).


OVCAR - 3 cell confluency by IncuCyte ® during 72h of treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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OVCAR-3 cell confluency by IncuCyte® during 72h of treatment with APR-246 +/- MK-571 (n = 3).


Growth suppression in HCT116 WT ( n = 4 ) and R248W cells ( n = 3 ) after 72h treatment with APR - 246 + / - MRP1 inhibitor Reversan , as shown by WST - 1 .

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Growth suppression in HCT116 WT (n = 4) and R248W cells (n = 3) after 72h treatment with APR-246 +/- MRP1 inhibitor Reversan, as shown by WST-1.


Western blot analysis of MRP1 ( Cell Signaling . ) , p53 ( DO - 1 ) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53 .

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Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53.


Growth suppression ( WST - 1 assay ) of HCT116 WT and R248W cells transfected with MRP1 siRNA ( n ≥ 5 and n ≥ 2 respectively ) after 48h APR - 246 treatment . Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs . Data and n of individual siRNAs are shown in Fig S1J , data is also part of Fig 7B .

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Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with MRP1 siRNA (n ≥ 5 and n ≥ 2 respectively) after 48h APR-246 treatment. Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs. Data and n of individual siRNAs are shown in Fig S1J, data is also part of Fig 7B.


Western blot analysis of MRP1 ( Cell Signaling . ) , p53 ( DO - 1 ) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector ( EV ) and MRP1 expression vector .

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Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector (EV) and MRP1 expression vector.


Growth suppression ( WST - 1 assay ) of HCT116 WT and R248W cells transfected with empty vector ( EV ) and MRP1 vector after 72h APR - 246 treatment ( n = 4 ) . Data is also part of Fig S7E .

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Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with empty vector (EV) and MRP1 vector after 72h APR-246 treatment (n = 4). Data is also part of Fig S7E.


Growth of Eso26 xenografts ( R248W mutant TP53 ) in mice treated with vehicle , APR - 246 ( 50mg / kg ) , MK - 571 ( 50mg / kg ) or APR - 246 and MK - 571 for 16 days . p values determined by one - way ANOVA with Tukey ' s correction at day 18 , n is indicated in the figure . Kaplan - Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle , APR - 246 ( 50mg / kg ) , MK - 571 ( 50mg / kg ) or APR - 246 and MK - 571 for 16 days . Mice that were culled at ethical endpoints prior to reaching a tumor volume of > 1400mm3 are marked as censored observations . p values were determined by Log - rank ( Mantell - Cox ) test between different treatment groups . n is indicated in the figure .

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

Growth of Eso26 xenografts (R248W mutant TP53) in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. p values determined by one-way ANOVA with Tukey's correction at day 18, n is indicated in the figure.   Kaplan-Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. Mice that were culled at ethical endpoints prior to reaching a tumor volume of >1400mm3 are marked as censored observations. p values were determined by Log-rank (Mantell-Cox) test between different treatment groups. n is indicated in the figure.


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 .

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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 .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 .

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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 ) .

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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 B-CONTROLLED_VAR 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-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

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

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 .

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 B-CONTROLLED_VAR 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

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 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

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 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

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

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 ) .

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

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 ) .

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

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 .

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

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 )

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

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 ) .

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

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

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(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 .

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(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 ) .

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(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.