PML - GFP HeLa Kyoto cells were lipofected with a cDNA encoding for mCherry - VHL . 24 h post - transfection , cells were left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Where indicated ( OP - puro + MG132 + CHX ) , translation was inhibited during stress with cycloheximide ( CHX ; 50 μg / ml ) . In the lower panel , cells were allowed to recover for 5 h in drug - free medium ( Control ) , or with VER ( 40 µM ) . The subcellular distribution of PML - GFP and mCherry - VHL was studied by live - cell confocal imaging . Representative pictures are shown . Scale bars : 5 µm . Quantitation of the fluorescence intensity recovery after bleach of PML - GFP . PML - GFP HeLa Kyoto overexpressing mCherry - VHL for 24 h and treated as described in ( A ) were analyzed . The mean of 19 - 23 FRAP curves and the fitting curves are shown in black and red respectively . In gray , the SD is shown .

B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

PML-GFP HeLa Kyoto cells were lipofected with a cDNA encoding for mCherry-VHL. 24 h post-transfection, cells were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Where indicated (OP-puro + MG132 + CHX), translation was inhibited during stress with cycloheximide (CHX; 50 μg/ml). In the lower panel, cells were allowed to recover for 5 h in drug-free medium (Control), or with VER (40 µM). The subcellular distribution of PML-GFP and mCherry-VHL was studied by live-cell confocal imaging. Representative pictures are shown. Scale bars: 5 µm. Quantitation of the fluorescence intensity recovery after bleach of PML-GFP. PML-GFP HeLa Kyoto overexpressing mCherry-VHL for 24 h and treated as described in (A) were analyzed. The mean of 19-23 FRAP curves and the fitting curves are shown in black and red respectively. In gray, the SD is shown.


Intranuclear distribution of 20S proteasomes , HSP70 , VCP and DRiPs in HeLa cells treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h and allowed to recover for 5 h in drug - free medium ( control ) , with VER ( 40 µM ) , Eeyarestatin I ( 5 µM ) or MG132 ( 10 µM ) . Scale bars : 5 µm .

O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

Intranuclear distribution of 20S proteasomes, HSP70, VCP and DRiPs in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h and allowed to recover for 5 h in drug-free medium (control), with VER (40 µM), Eeyarestatin I (5 µM) or MG132 (10 µM). Scale bars: 5 µm.


GFP - PSMA7 HeLa Kyoto cells were left untreated or treated as described in A . Cells were subjected to FRAP to analyze GFP - PSMA7 mobility . Quantitation of three populations of GFP - PSMA7 molecules is shown : fast moving , slow moving and immobile ; statistical significance via One - way ANOVA ; n = 12 - 14 ± SEM .

B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

GFP-PSMA7 HeLa Kyoto cells were left untreated or treated as described in A. Cells were subjected to FRAP to analyze GFP-PSMA7 mobility. Quantitation of three populations of GFP-PSMA7 molecules is shown: fast moving, slow moving and immobile; statistical significance via One-way ANOVA; n = 12 - 14 ± SEM.


Subcellular distribution of 53BP1 and DRiPs in HeLa cells that were left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h , followed by 5h recovery in drug - free medium ( + rec . control ) , with VER ( 40 µM ; + rec . VER ) or Eeyarestatin I ( 5 µM ; + rec . EeyI ) . Where indicated , cells were treated with MG132 , VER or EeyI alone for 5 h . Scale bars : 20 µm . Quantitation of the % of cells with 53BP1 foci . Cells were treated as described in ( A ) and divided in three categories , based on the number of 53BP1 foci / nucleus : 0 , 1 - 2 and > 3 . Number of cells counted / condition : 1112 - 1537 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 01 .

O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O 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-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

Subcellular distribution of 53BP1 and DRiPs in HeLa cells that were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h, followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI). Where indicated, cells were treated with MG132, VER or EeyI alone for 5 h. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (A) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 1112 - 1537 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.


HeLa cells were lipofected with cDNAs encoding for HA - Ubiquitin , Flag - Ubiquitin or Flag - VCP 24 h post - transfection , cells were either left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) . Cells were processed for staining of HA , Flag and endogenous 53BP1 . * indicates cells that do not express HA - or Flag - Ubiquitin and show no rescue of 53BP1 foci formation after stress . Scale bars : 20 µm . Quantitation of the % of cells with 53BP1 foci . Cells were treated as described in ( C ) and divided in three categories , based on the number of 53BP1 foci / nucleus : 0 , 1 - 2 and > 3 . Number of cells counted / condition : 710 - 938 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 01 .

B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells were lipofected with cDNAs encoding for HA-Ubiquitin, Flag-Ubiquitin or Flag-VCP 24 h post-transfection, cells were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM). Cells were processed for staining of HA, Flag and endogenous 53BP1. * indicates cells that do not express HA- or Flag-Ubiquitin and show no rescue of 53BP1 foci formation after stress. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (C) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 710 - 938 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.


Colony formation assay . The number of colonies surviving 10 days after plating is shown . Control cells or cells treated with MG132 ( 10 µM ) and puromycin ( 5 µg / ml ) , followed by 5h recovery in drug - free medium ( + rec . control ) , with VER ( 40 µM ; + rec . VER ) or Eeyarestatin I ( 5 µM ; + rec . EeyI ) are shown . Where indicated , cells were treated with VER or EeyI alone for 5 h . Number of colonies counted / condition : up to 526 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 03 , ± s . e . m .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colony formation assay. The number of colonies surviving 10 days after plating is shown. Control cells or cells treated with MG132 (10 µM) and puromycin (5 µg/ml), followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI) are shown. Where indicated, cells were treated with VER or EeyI alone for 5 h. Number of colonies counted/condition: up to 526 in three independent experiments; statistical significance via One-way ANOVA; p < 0.03, ± s.e.m.


( b ) SAR405 inhibits the function of Vps34 in the GFP - FYVE cellular assay . HeLa cells stably transfected with GFP - FYVE were incubated with DMSO ( left ) or with 1 μM SAR405 ( right ) for 2 h . Cells were fixed , and nuclei were stained using Hoechst 33342 ( blue ) . Fluorescence was analyzed using an imaging cytometer ( X40 ) . One representative image out of three independent experiments is depicted . Scale bars , 20 μm .

O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(b) SAR405 inhibits the function of Vps34 in the GFP-FYVE cellular assay. HeLa cells stably transfected with GFP-FYVE were incubated with DMSO (left) or with 1 μM SAR405 (right) for 2 h. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). Fluorescence was analyzed using an imaging cytometer (X40). One representative image out of three independent experiments is depicted. Scale bars, 20 μm.


( c ) SAR405 does not prevent phosphorylation of Akt and S6 . U87MG cells were treated with SAR405 or PI103 for 2 h at the indicated concentrations . The effect of compounds on phosphorylated Akt ( p - Akt ) Ser473 and Thr308 residues , total Akt and on phosphorylated S6 ( Ser240 and Ser244 residues ) and total S6 protein was measured using western blotting . One representative experiment from at least two independent experiments is shown . Full - length blots are shown in Supplementary Figure 6 .

O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY 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) SAR405 does not prevent phosphorylation of Akt and S6. U87MG cells were treated with SAR405 or PI103 for 2 h at the indicated concentrations. The effect of compounds on phosphorylated Akt (p-Akt) Ser473 and Thr308 residues, total Akt and on phosphorylated S6 (Ser240 and Ser244 residues) and total S6 protein was measured using western blotting. One representative experiment from at least two independent experiments is shown. Full-length blots are shown in Supplementary Figure 6.


( a ) SAR405 induces late endosome - lysosome swelling . RKO cells were treated with DMSO ( upper panels ) or with 10 μM SAR405 for 16 h ( lower panels ) . The late endosomes - lysosomes compartment was assessed using Lysotracker green ( left ) or Alexa 488 - conjugated anti - Lamp1 antibodies ( right ) , as indicated . Nuclei were stained using Hoechst 33342 ( blue ) . One representative image chosen from three independent experiments , showing fluorescence imaged using a Zeiss microscope ( X40 ) , is shown for each condition . Arrows indicate accumulation of swollen late endosome - lysosomes . Scale bars , 20 μm .

O O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O

(a) SAR405 induces late endosome-lysosome swelling. RKO cells were treated with DMSO (upper panels) or with 10 μM SAR405 for 16 h (lower panels). The late endosomes-lysosomes compartment was assessed using Lysotracker green (left) or Alexa 488-conjugated anti-Lamp1 antibodies (right), as indicated. Nuclei were stained using Hoechst 33342 (blue). One representative image chosen from three independent experiments, showing fluorescence imaged using a Zeiss microscope (X40), is shown for each condition. Arrows indicate accumulation of swollen late endosome-lysosomes. Scale bars, 20 μm.


( b ) SAR405 triggers accumulation of p62 . RKO cells were treated with the indicated concentration of Vps34 inhibitor for 24 h . The effect on the p62 biomarker was analyzed by western blotting . GAPDH was used as a loading control . The results are representative of at least two independent experiments . Full - length blots are shown in Supplementary Figure 14 .

O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O 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) SAR405 triggers accumulation of p62. RKO cells were treated with the indicated concentration of Vps34 inhibitor for 24 h. The effect on the p62 biomarker was analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 14.


( c ) SAR405 affects the maturation of cathepsin D ( cat D ) . RKO cells were treated at the indicated concentrations of SAR405 for 24 h . The different forms of cathepsin D ( PPCD , pre - pro - catD ) were analyzed by western blotting . GAPDH was used as a loading control . The results are representative of at least two independent experiments . Full - length blots are shown in Supplementary Figure 16 .

O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O 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) SAR405 affects the maturation of cathepsin D (cat D). RKO cells were treated at the indicated concentrations of SAR405 for 24 h. The different forms of cathepsin D (PPCD, pre-pro-catD) were analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 16.


( a ) GFP - LC3 HeLa cells were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine and treated with Vps34 inhibitor . Cells were fixed , and nuclei were stained using Hoechst 33342 ( blue ) . One representative image , acquired with an iCyte imaging cytometer , for three independent experiments is depicted ( X40 ) . White arrows indicate GFP - LC3 spots , which correspond to the autophagosomes . Scale bars , 10 μm . ( b ) Histograms show the percentage of positive cells and GFP intensity , measured using an imaging cytometer for different compound concentrations on GFP - LC3 HeLa cells . Black and gray histograms correspond to fed and starved conditions , respectively . For GFP intensity , the values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments .

O O O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O 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) GFP-LC3 HeLa cells were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine and treated with Vps34 inhibitor. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image, acquired with an iCyte imaging cytometer, for three independent experiments is depicted (X40). White arrows indicate GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells and GFP intensity, measured using an imaging cytometer for different compound concentrations on GFP-LC3 HeLa cells. Black and gray histograms correspond to fed and starved conditions, respectively. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.


( c ) Wild - type HeLa and H1299 cell lines were cultured either in fed or starved conditions with EBSS and 10 μM hydroxychloroquine ( HCQ ) , as indicated . Cells were treated at the same time with SAR405 at different concentrations . LC3 - II was measured by western blotting , and GAPDH was used as a loading control . One representative experiment for three independent experiments is shown . Full - length blots are shown in Supplementary Figure 18 . ( d ) Histograms show the LC3 - II / GAPDH ratio for each condition in the two cell lines . Black and gray histograms correspond to fed condition and EBSS conditions , respectively . The addition of hydroxychloroquine at 10 μM is indicated . Values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments .

O O O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O 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-SMALL_MOLECULE O O O O 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) Wild-type HeLa and H1299 cell lines were cultured either in fed or starved conditions with EBSS and 10 μM hydroxychloroquine (HCQ), as indicated. Cells were treated at the same time with SAR405 at different concentrations. LC3-II was measured by western blotting, and GAPDH was used as a loading control. One representative experiment for three independent experiments is shown. Full-length blots are shown in Supplementary Figure 18. (d) Histograms show the LC3-II/GAPDH ratio for each condition in the two cell lines. Black and gray histograms correspond to fed condition and EBSS conditions, respectively. The addition of hydroxychloroquine at 10 μM is indicated. Values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.


( a ) GFP - LC3 H1299 cells were treated with 1 μM of the catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO ( left ) or with 1 μM SAR405 ( right ) . Cells were fixed , and nuclei were stained using Hoechst 33342 ( blue ) . One representative image of cells out of three independent experiments , acquired using an iCyte imaging cytometer , is shown ( X40 ) . White arrows indicate the GFP - LC3 spots , which correspond to the autophagosomes . Scale bars , 10 μm . ( b ) Histograms show the percentage of positive cells ( top ) and the GFP intensity ( bottom ) , as measured using an imaging cytometer . Treatment of GFP - LC3 H1299 in fed conditions with 1 μM of AZD8055 and different concentrations of SAR405 is indicated . For GFP intensity , the values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments .

O O O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O 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) GFP-LC3 H1299 cells were treated with 1 μM of the catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO (left) or with 1 μM SAR405 (right). Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image of cells out of three independent experiments, acquired using an iCyte imaging cytometer, is shown (X40). White arrows indicate the GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells (top) and the GFP intensity (bottom), as measured using an imaging cytometer. Treatment of GFP-LC3 H1299 in fed conditions with 1 μM of AZD8055 and different concentrations of SAR405 is indicated. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.


( c ) The concentration needed to reach 40 % inhibition ( IC40 ) of cell proliferation is indicated . The top histograms indicate the IC40 of SAR405 as a single agent and in combination with everolimus in the three cell lines . The bottom histograms indicate the IC40 of everolimus as a single agent and in combination with SAR405 . The IC40 values for the combination of the two compounds were determined using rays with an effective fraction ∼ 0 . 5 , corresponding to compounds that are in equipotent proportion in the mixture ( Supplementary Fig . 20 ) . The absolute IC40 determination of both compounds is represented as the geometric mean from two or 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 O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The concentration needed to reach 40% inhibition (IC40) of cell proliferation is indicated. The top histograms indicate the IC40 of SAR405 as a single agent and in combination with everolimus in the three cell lines. The bottom histograms indicate the IC40 of everolimus as a single agent and in combination with SAR405. The IC40 values for the combination of the two compounds were determined using rays with an effective fraction ∼0.5, corresponding to compounds that are in equipotent proportion in the mixture (Supplementary Fig. 20). The absolute IC40 determination of both compounds is represented as the geometric mean from two or three independent experiments.


D Cruciform unfolding assay with increasing WRN concentrations and cruciform consisting of random inverted repeats ( random - IR ) . Top , quantitation of cruciform unfolding . The amount of linear DNA from the " No protein " lane was subtracted from all other samples . Averages shown ; n = 3 technical replicates ; error bars , SEM .

O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Cruciform unfolding assay with increasing WRN concentrations and cruciform consisting of random inverted repeats (random-IR). Top, quantitation of cruciform unfolding. The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n = 3 technical replicates; error bars, SEM.


E Cruciform unfolding assays as in ( D ) with WRN , WRN K577M , and WRN E84A ( all used at 10 nM ) , carried out either in the presence or absence of ATP , or with ATP - ƔS . Averages shown ; n ≥ 3 technical replicates ; error bars , SEM .

O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O

E Cruciform unfolding assays as in (D) with WRN, WRN K577M, and WRN E84A (all used at 10 nM), carried out either in the presence or absence of ATP, or with ATP-ƔS. Averages shown; n ≥ 3 technical replicates; error bars, SEM.


G Representative cruciform unfolding assays with increasing concentrations of WRN , together with human RPA ( hRPA , 30 nM ) , yeast S . cerevisiae RPA ( yRPA , 30 nM ) , or human mitochondrial SSB ( hmtSSB , 50 nM ) . pUC19 with the random - IR cruciform structure was used as a substrate . H Quantitation of assays such as in ( G ) . The amount of linear DNA from the " No protein " lane was subtracted from all other samples . Averages shown ; n ≥ 3 technical replicates ; error bars , SEM .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-ORGANISM B-GENEPROD O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O B-ORGANISM B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative cruciform unfolding assays with increasing concentrations of WRN, together with human RPA (hRPA, 30 nM), yeast S. cerevisiae RPA (yRPA, 30 nM), or human mitochondrial SSB (hmtSSB, 50 nM). pUC19 with the random-IR cruciform structure was used as a substrate. H Quantitation of assays such as in (G). The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n ≥ 3 technical replicates; error bars, SEM.


A Representative assays with cruciform DNA ( consisting of random - IR ) either without or with WRN incubation prior to cruciform detection by SLX1 - SLX4CCD . The ScaI restriction site is 931 bp away from the cruciform site cleaved by SLX1 - SLX4CCD . Combined activity of ScaI and SMX results in 931 and 1795 bp bands indicated by the green arrows . The intensity of these bands decreases upon WRN incubation , resulting predominantly in linear DNA cut by ScaI , suggesting that WRN removes the substrate for SLX1 - SLX4CCD . The catalytic dead version of SLX1 - SLX4CCD ( SLX1CD - SLX4CCD ) , where SLX1 carries an R41A mutations in its nuclease domain , has no activity on the cruciform DNA . B Quantitation of assays such as in ( A ) . Averages shown ; n = 3 technical replicates ; error bars , SEM .

O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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 Representative assays with cruciform DNA (consisting of random-IR) either without or with WRN incubation prior to cruciform detection by SLX1-SLX4CCD. The ScaI restriction site is 931 bp away from the cruciform site cleaved by SLX1-SLX4CCD. Combined activity of ScaI and SMX results in 931 and 1795 bp bands indicated by the green arrows. The intensity of these bands decreases upon WRN incubation, resulting predominantly in linear DNA cut by ScaI, suggesting that WRN removes the substrate for SLX1-SLX4CCD. The catalytic dead version of SLX1-SLX4CCD (SLX1CD-SLX4CCD), where SLX1 carries an R41A mutations in its nuclease domain, has no activity on the cruciform DNA. B Quantitation of assays such as in (A). Averages shown; n = 3 technical replicates; error bars, SEM.


B Mapping of TA repeats cruciform structure . T7 Endonuclease I activity , followed by SspI , cleaves the cruciform within negative supercoiled DNA , producing two bands of the expected size ( 607 bp and 2119 bp , indicated by the green arrows , lane 2 ) . Simultaneous incubation of the substrate with SspI and EcoRI allows EcoRI cutting at the TA repeats sequence ( lane 5 ) , while the extruded cruciform is refractory to EcoRI digestion ( lane 4 , the * indicates scDNA ) .

O O O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O

B Mapping of TA repeats cruciform structure. T7 Endonuclease I activity, followed by SspI, cleaves the cruciform within negative supercoiled DNA, producing two bands of the expected size (607 bp and 2119 bp, indicated by the green arrows, lane 2). Simultaneous incubation of the substrate with SspI and EcoRI allows EcoRI cutting at the TA repeats sequence (lane 5), while the extruded cruciform is refractory to EcoRI digestion (lane 4, the * indicates scDNA).


C Representative cruciform detection assays . Plasmid DNA was subjected to Cruciform extrusion procedure , where indicated . Supercoiled DNA ( cruciform , refractory to EcoRI ) and linear DNA ( no cruciform , cleaved by EcoRI ) were resolved on a 1 % agarose gel , stained with GelRed . Top , quantitation of extruded cruciform in % with respect to the amount of total DNA that is present in the lane . Averages shown ; n = 4 technical replicates ; error bars , SEM . D Representative cruciform unfolding assays with increasing WRN concentrations , using pUC19 with either random inverted repeats or TA repeats as a substrate . Top , quantitation of cruciform unfolding . The amount of linear DNA from the " No protein " lane was subtracted from all other samples . Averages shown ; n = 3 technical replicates ; error bars , SEM .

O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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 Representative cruciform detection assays. Plasmid DNA was subjected to Cruciform extrusion procedure, where indicated. Supercoiled DNA (cruciform, refractory to EcoRI) and linear DNA (no cruciform, cleaved by EcoRI) were resolved on a 1% agarose gel, stained with GelRed. Top, quantitation of extruded cruciform in % with respect to the amount of total DNA that is present in the lane. Averages shown; n = 4 technical replicates; error bars, SEM. D Representative cruciform unfolding assays with increasing WRN concentrations, using pUC19 with either random inverted repeats or TA repeats as a substrate. Top, quantitation of cruciform unfolding. The amount of linear DNA from the "No protein" lane was subtracted from all other samples. Averages shown; n = 3 technical replicates; error bars, SEM.


A Recombinant BLM and S . cerevisiae Sgs1 used in this study . The gel was stained with Coomassie Brilliant blue .

O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

A Recombinant BLM and S. cerevisiae Sgs1 used in this study. The gel was stained with Coomassie Brilliant blue.


B Representative cruciform unfolding assay with increasing concentrations of WRN , BLM , and Sgs1 . Cruciform DNA consisting of random inverted repeats was used . C Quantitation of assays such as ( B ) . Averages shown ; n ≥ 3 technical replicates ; error bars , SEM .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O 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 Representative cruciform unfolding assay with increasing concentrations of WRN, BLM, and Sgs1. Cruciform DNA consisting of random inverted repeats was used. C Quantitation of assays such as (B). Averages shown; n ≥ 3 technical replicates; error bars, SEM.


F Representative cruciform unfolding assays with increasing concentrations of WRN and WRN - 3A . Cruciform DNA consisting of random inverted repeats was used . G Quantitation of assays such as in ( F ) . Averages shown ; n = 3 technical replicates ; error bars , SEM .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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 Representative cruciform unfolding assays with increasing concentrations of WRN and WRN-3A. Cruciform DNA consisting of random inverted repeats was used. G Quantitation of assays such as in (F). Averages shown; n = 3 technical replicates; error bars, SEM.


B Recombinant full - length and truncation WRN variants used in this study are shown . The gel was stained with Coomassie Brilliant blue .

O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

B Recombinant full-length and truncation WRN variants used in this study are shown. The gel was stained with Coomassie Brilliant blue.


D Cruciform unfolding assays with increasing concentrations of WRN variants . E Quantitation of assays such as shown in ( D ) . Averages shown ; n = 3 technical replicates ; error bars , SEM .

O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Cruciform unfolding assays with increasing concentrations of WRN variants. E Quantitation of assays such as shown in (D). Averages shown; n = 3 technical replicates; error bars, SEM.


A MutL homologue heterodimers used in this study : MutLα ( MLH1 - PMS2 ) , MutLβ ( MLH1 - PMS1 ) and MutLγ ( MLH1 - MLH3 ) . B MutS homologue heterodimers used in this study : MutSα ( MSH2 - MSH6 ) , MutSβ ( MSH2 - MSH3 ) and MutSγ ( MSH4 - MSH5 ) .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O

A MutL homologue heterodimers used in this study: MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1) and MutLγ (MLH1-MLH3). B MutS homologue heterodimers used in this study: MutSα (MSH2-MSH6), MutSβ (MSH2-MSH3) and MutSγ (MSH4-MSH5).


E Representative electrophoretic mobility shift assays with the MutS and MutL homologue heterodimers , using an oligonucleotide - based Holliday junction as a substrate . Red asterisk indicates the position of the radioactive label .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O 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 Representative electrophoretic mobility shift assays with the MutS and MutL homologue heterodimers, using an oligonucleotide-based Holliday junction as a substrate. Red asterisk indicates the position of the radioactive label.


F Representative cruciform unfolding assays with MutSβ and MutS ⍺ , carried out without or with ATP , using the random - IR cruciform as a substrate . Top , quantitation of cruciform unfolding . Averages shown ; n = 3 technical replicates ; error bars , SEM . G Representative cruciform unfolding assays with MutSβ and MutS ⍺ , carried out without or with hRPA , using the random - IR cruciform as a substrate . Top , quantitation of cruciform unfolding . Averages shown ; n = 3 technical replicates ; error bars , SEM .

O O O B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O

F Representative cruciform unfolding assays with MutSβ and MutS⍺, carried out without or with ATP, using the random-IR cruciform as a substrate. Top, quantitation of cruciform unfolding. Averages shown; n = 3 technical replicates; error bars, SEM. G Representative cruciform unfolding assays with MutSβ and MutS⍺, carried out without or with hRPA, using the random-IR cruciform as a substrate. Top, quantitation of cruciform unfolding. Averages shown; n = 3 technical replicates; error bars, SEM.


A Representative polyacrylamide gel showing recombinant MutSβ , MutSβ - 3K255A ( MSH3 with mutation K255A , deficient in mismatch recognition ) and MutSβ - 3K902A ( MSH3 with mutation K902A , ATPase - deficient ) . The gel was stained with Coomassie Brilliant blue .

O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

A Representative polyacrylamide gel showing recombinant MutSβ, MutSβ-3K255A (MSH3 with mutation K255A, deficient in mismatch recognition) and MutSβ-3K902A (MSH3 with mutation K902A, ATPase-deficient). The gel was stained with Coomassie Brilliant blue.


B Electrophoretic mobility shift assay with MutSβ , MutSβ - 3K255A and MutSβ - 3K902A , using either dsDNA ( 50 bp ) or dsDNA bearing one extrahelical T , as a substrate . 6 % native acrylamide gel was used .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

B Electrophoretic mobility shift assay with MutSβ, MutSβ-3K255A and MutSβ-3K902A, using either dsDNA (50 bp) or dsDNA bearing one extrahelical T, as a substrate. 6% native acrylamide gel was used.


G Unfolding of TA cruciform by WRN and MMR proteins . Bottom , representative experiments ; top , quantitation ; averages shown ; n = 4 technical replicates ; error bars , SEM . H Unfolding of TA cruciform by MutLα and wild - type WRN or helicase dead WRN - K577M . Bottom , representative experiments ; top , quantitation , averages shown ; n = 4 technical replicates ; error bars , SEM .

O B-EXP_ASSAY O O O O B-GENEPROD O O O O 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-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O 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 Unfolding of TA cruciform by WRN and MMR proteins. Bottom, representative experiments; top, quantitation; averages shown; n = 4 technical replicates; error bars, SEM. H Unfolding of TA cruciform by MutLα and wild-type WRN or helicase dead WRN-K577M. Bottom, representative experiments; top, quantitation, averages shown; n = 4 technical replicates; error bars, SEM.


( A ) Binding of wild type DUBm - Ubp8 to monoubiquitin . ( B ) Binding of DUBmUbp8C146A to monoubiquitin . ( C ) Binding of DUBm - Ubp8C146S to monoubiquitin . ( D ) Binding of DUBm - Ubp8C146R to monoubiquitin . ( E ) Binding of DUBm - Ubp8C146A to K48 diubiquitin . ( F ) Binding of DUBm - Ubp8C146S to K48 diubiquitin .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-SMALL_MOLECULE O

(A) Binding of wild type DUBm-Ubp8 to monoubiquitin. (B) Binding of DUBmUbp8C146A to monoubiquitin. (C) Binding of DUBm-Ubp8C146S to monoubiquitin. (D) Binding of DUBm-Ubp8C146R to monoubiquitin. (E) Binding of DUBm-Ubp8C146A to K48 diubiquitin. (F) Binding of DUBm-Ubp8C146S to K48 diubiquitin.


Binding was measured by fluorescence polarization using N - terminally TAMRA - labeled monoubiquitin . The dissociation constants for ubiquitin binding to USP4 WT and C311A are 92 ± 21 nM ( Clerici et . al , 2014 ) and 0 . 60 ± 0 . 17 nM , respectively . Error bars are s . d . calculated on five measurements per point .

B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Binding was measured by fluorescence polarization using N-terminally TAMRA-labeled monoubiquitin. The dissociation constants for ubiquitin binding to USP4 WT and C311A are 92 ± 21 nM (Clerici et. al, 2014) and 0.60 ± 0.17 nM, respectively. Error bars are s.d. calculated on five measurements per point.


( A ) Equilibrium binding of OTUD1 C320A and C320R to K63 - linked diubiquitin was measured by fluorescence polarization using FlAsH tagged K63 - linked diubiquitin in which the proximal ubiquitin was fluorescently - labeled . Error bars indicate s . d . and are based on three measurements per data point . One representative experiment of two is shown .

O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O 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) Equilibrium binding of OTUD1 C320A and C320R to K63-linked diubiquitin was measured by fluorescence polarization using FlAsH tagged K63-linked diubiquitin in which the proximal ubiquitin was fluorescently-labeled. Error bars indicate s.d. and are based on three measurements per data point. One representative experiment of two is shown.


( B ) Whole cell lysates of HEK293 cells expressing HA - tagged OTUD1 WT , C320R , and C320A were immunoblotted with indicated antibodies . One representative experiment of three is shown .

O O O O O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

(B) Whole cell lysates of HEK293 cells expressing HA-tagged OTUD1 WT, C320R, and C320A were immunoblotted with indicated antibodies. One representative experiment of three is shown.


Polyubiquitin chains were co - immunoprecipitated with FLAG - PSMD4 , an ubiquitin receptor for the 26S proteasome , from cells expressing either USP14 wild - type , C114A , or C114R . One representative experiment of two is shown .

B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

Polyubiquitin chains were co-immunoprecipitated with FLAG-PSMD4, an ubiquitin receptor for the 26S proteasome, from cells expressing either USP14 wild-type, C114A, or C114R. One representative experiment of two is shown.


Human derived GBM tumors were processed as in ( A ) . Representative western blot were performed ( F ) and the percentage of ER - lumenal protein cytosolic localization ( G ) were quantified . Data are the average from six different tumors . N = 6 Bars and error bars indicate

B-ORGANISM O B-DISEASE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

Human derived GBM tumors were processed as in (A). Representative western blot were performed (F) and the percentage of ER-lumenal protein cytosolic localization (G) were quantified. Data are the average from six different tumors. N=6 Bars and error bars indicate mean


Left : Representative images of cells were transfected with super - folder GFP ( sfGFP ) treated with 250ng / mL Tunicamycin ( Tm ) , 50nM Thapsigargin ( Tg ) and 250ng / mL Brefeldin - A ( BFA ) for 24 hours . Right : Quantification of the microscopy images of cells expressing ER - targeted sfGFP and the cytosolically localized mCherry . Data values are the mean ± SD of technical replicates ( n = 10 ) from 3 independent experiments ( * * * * p < 0 . 0001 ) . One - way ANOVA was applied for the statistical analysis through the GraphPad Prism 9 software . Scale bar 15μm .

O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Left: Representative images of cells were transfected with super-folder GFP (sfGFP) treated with 250ng/mL Tunicamycin (Tm), 50nM Thapsigargin (Tg) and 250ng/mL Brefeldin-A (BFA) for 24 hours. Right: Quantification of the microscopy images of cells expressing ER-targeted sfGFP and the cytosolically localized mCherry. Data values are the mean ± SD of technical replicates (n=10) from 3 independent experiments (****p<0.0001). One-way ANOVA was applied for the statistical analysis through the GraphPad Prism 9 software. Scale bar 15μm.


Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin ( NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer ) protocols , representative western blots are showed .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O

Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) protocols, representative western blots are showed.


Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin ( NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer ) or differential centrifugation protocols , representative western blots are showed .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O

Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) or differential centrifugation protocols, representative western blots are showed.


Subcellular protein fractionation of several ER resident proteins in ( A ) GL261 , ( B ) U87 and ( C ) A549 cells treated with the indicated concentrations of Tm or Tg using Digitonin . ( NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer ) Quantification of the subcellular protein fractionation of several ER endogenous proteins in GL261 , U87 and A549 cells as in ( A - C ) respectively . Biological triplicates , mean ± SD calculated using Prism 9 ( GraphPad ) .

O O O O O B-SUBCELLULAR O O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O

Subcellular protein fractionation of several ER resident proteins in (A) GL261, (B) U87 and (C) A549 cells treated with the indicated concentrations of Tm or Tg using Digitonin. (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) Quantification of the subcellular protein fractionation of several ER endogenous proteins in GL261, U87 and A549 cells as in (A-C) respectively. Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad).


Representative transmission electron microscopy images of the gold particles distribution ( after immunogold labelling with PDIA3 antibodies ) in A549 cells treated with DMSO or Tm . In the inserts , gold particles in the ER were surrounded by red circles and those in the rest of the cytoplasm by blue circles . ( Scale bar 500 nm ) . n represents the nucleus and m represents the mitochondria . ( C ) violin plots of the gold particles distribution from the electron microscopy experiment ( immunogold labelling of PDIA3 ) as shown in Figure 4B and 4C . * P - value = 0 . 0486 . n = 8 for DMSO and n = 8 for Tm . Thick horizontal lines represent mean ± SD - lighter dashed lines . Differences were analyzed by Unpaired Student ' s t - test using Prism 9 ( GraphPad ) ,

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative transmission electron microscopy images of the gold particles distribution (after immunogold labelling with PDIA3 antibodies) in A549 cells treated with DMSO or Tm. In the inserts, gold particles in the ER were surrounded by red circles and those in the rest of the cytoplasm by blue circles. (Scale bar 500 nm). n represents the nucleus and m represents the mitochondria. (C) violin plots of the gold particles distribution from the electron microscopy experiment (immunogold labelling of PDIA3) as shown in Figure 4B and 4C. *P-value=0.0486. n=8 for DMSO and n=8 for Tm. Thick horizontal lines represent mean ± SD -lighter dashed lines. Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad),


Immunoprecipitation of p53 and AGR2 in the digitonin fraction of A549 cells . A549 cells were treated with Tunicamycin ( Tm 100ng / ml ) , Thapsigargin ( Tg 25nM ) and Brefeldin A ( BFA 0 , 1 and 0 , 25nM ) . Endogenous p53 was immunoprecipitated from the cytoplasmic fraction ( digitonin fraction ) of A549 cells . Coprecipitated endogenous AGR2 was detected by western blot .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_LINE O O B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O B-CELL_LINE O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

Immunoprecipitation of p53 and AGR2 in the digitonin fraction of A549 cells. A549 cells were treated with Tunicamycin (Tm 100ng/ml), Thapsigargin (Tg 25nM) and Brefeldin A (BFA 0,1 and 0,25nM). Endogenous p53 was immunoprecipitated from the cytoplasmic fraction (digitonin fraction) of A549 cells. Coprecipitated endogenous AGR2 was detected by western blot.


A549 were treated with Etoposide ( Eto ) for 2 hours to induce p53 pathway . Then cells have been challenged with Tm , Tg and BFA at the indicated concentrations for 16 hours . Luciferase experiments were performed after 24 hours of transfection . Graph shows the fold induction of p53 luciferase construct . Western blot experiments for phospho - p53 , pan - p53 , p21 and HSP90 were performed as control . n = 3 , biological replicates ( mean ± SD ; Differences were analyzed by Unpaired Student ' s t - test using Prism 9 ( GraphPad ) , except when otherwise indicated . * p < 0 . 05 , * * * p < 0 . 001 .

B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A549 were treated with Etoposide (Eto) for 2 hours to induce p53 pathway. Then cells have been challenged with Tm, Tg and BFA at the indicated concentrations for 16 hours. Luciferase experiments were performed after 24 hours of transfection. Graph shows the fold induction of p53 luciferase construct. Western blot experiments for phospho-p53, pan-p53, p21 and HSP90 were performed as control. n=3, biological replicates (mean ± SD; Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated. *p<0.05, ***p < 0.001.


A549 were transfected with control siRNA ( siC ) and AGR2 - tergetd siRNA ( siAGR2 ) . After 24 hours , cells were transfected with p53 - luciferase construct . Cells were then treated as in ( C ) and luciferase experiments were performed . n = 4 , biological replicates ( mean ± SD . Differences were analyzed by Unpaired Student ' s t - test using Prism 9 ( GraphPad ) , except when otherwise indicated .

B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A549 were transfected with control siRNA (siC) and AGR2-tergetd siRNA (siAGR2). After 24 hours, cells were transfected with p53-luciferase construct. Cells were then treated as in (C) and luciferase experiments were performed. n=4, biological replicates (mean ± SD. Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated.


Left : Western blot experiments for phospho - p53 , pan - p53 , and p21 in AGR2 - silenced A549 cells exposed to ER stressors : 100ng / ml Tunicamycin ( Tm ) , 25nM Thapsigargin ( Tg ) and 0 . 25 nM Brefeldin A ( BFA ) , in the presence and absence of etoposide . Right : Quantification of the western blot . n = 3 , biological replicates , mean ± SD .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

Left: Western blot experiments for phospho-p53, pan-p53, and p21 in AGR2-silenced A549 cells exposed to ER stressors: 100ng/ml Tunicamycin ( Tm), 25nM Thapsigargin (Tg) and 0.25 nM Brefeldin A (BFA), in the presence and absence of etoposide. Right: Quantification of the western blot. n=3, biological replicates, mean ±SD.


A549 were transfected with the indicated constructs of differently targeted nanobodies , cells were then treated with Tunicamycin ( Tm 100 ng / ml and 250 ng / ml ) , Thapsigargin ( Tg 25 nM ) and Brefeldin A ( BFA 0 , 25 nM ) for 16 hours at the indicated concentration . Western blot experiments were performed and HSP90 were used as loading control . n = 3 , biological replicates ( mean ± SD of n = 3 independent experiments ( * * * p < 0 , 001 and * * p < 0 , 01 ) . Differences were analyzed by Unpaired Student ' s t - test using Prism 9 ( GraphPad ) , except when otherwise indicated .

B-CELL_LINE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A549 were transfected with the indicated constructs of differently targeted nanobodies, cells were then treated with Tunicamycin (Tm 100 ng/ml and 250 ng/ml), Thapsigargin (Tg 25 nM) and Brefeldin A (BFA 0,25 nM) for 16 hours at the indicated concentration. Western blot experiments were performed and HSP90 were used as loading control. n=3, biological replicates (mean ± SD of n=3 independent experiments (***p<0,001 and **p<0,01). Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated.


( A ) Western blot analysis of CRIP1 protein expression in 16 paired HCC tumor tissues ( T ) and adjacent nontumor tissues ( N ) . The graph represents CRIP1 protein expression normalized to GAPDH levels ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , paired ( A , Student ' s t -

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of CRIP1 protein expression in 16 paired HCC tumor tissues (T) and adjacent nontumor tissues (N). The graph represents CRIP1 protein expression normalized to GAPDH levels (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, paired (A, Student's t-test


( B ) Representative IHC images of CRIP1 protein expression in 108 paired HCC tumor tissues and adjacent nontumor tissues . Scale bars : 20 μm . Statistical analysis of immunohistochemical score of CRIP1 expression in HCC tumor tissues and paired adjacent tissues ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , paired B ) Student ' s t -

O O O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative IHC images of CRIP1 protein expression in 108 paired HCC tumor tissues and adjacent nontumor tissues. Scale bars: 20 μm. Statistical analysis of immunohistochemical score of CRIP1 expression in HCC tumor tissues and paired adjacent tissues (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, paired B) Student's t-test


( C ) CRIP1 mRNA expression in the TCGA - LIHC dataset . ( D ) mRNA levels of CRIP1 in the GSE36376 dataset from the GEO database . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , unpaired Student ' s t -

O O O B-GENEPROD O B-EXP_ASSAY O O O O B-DISEASE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CRIP1 mRNA expression in the TCGA-LIHC dataset. (D) mRNA levels of CRIP1 in the GSE36376 dataset from the GEO database. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, unpaired Student's t-test


The protein ( F ) levels of CRIP1 in HCC cell lines and normal LO2 hepatocytes . ( G ) Relative CRIP1 protein level compared to normal LO2 hepatocytes . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using a two - tailed , unpaired G ) Student ' s t -

O O O O O O O B-GENEPROD O B-DISEASE O O O O B-CELL_LINE B-CELL_TYPE O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The protein (F) levels of CRIP1 in HCC cell lines and normal LO2 hepatocytes. (G) Relative CRIP1 protein level compared to normal LO2 hepatocytes. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, unpaired G) Student's t-test


( H - I ) Kaplan - Meier survival analyses of OS ( H ) and PFS ( I ) based on CRIP1 expression levels in 108 HCC patients . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Differences were tested using log - rank test ( H - I ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-I) Kaplan-Meier survival analyses of OS (H) and PFS (I) based on CRIP1 expression levels in 108 HCC patients. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using log-rank test (H-I).


( B ) Tumor sphere formation assay in CRIP1 - overexpressing Huh7 and Hep3B cells . The data are shown as the relative fold change spheres compared with the vector group ( right ) . Scale bar : 50 μm . Data information : Data represent the mean ± SD at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t - test ( B ,

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tumor sphere formation assay in CRIP1-overexpressing Huh7 and Hep3B cells. The data are shown as the relative fold change spheres compared with the vector group (right). Scale bar: 50 μm. Data information: Data represent the mean ± SD at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test (B,


( C ) Western blot analysis of HCC cancer stem cell markers in CRIP1 - overexpressing Huh7 and Hep3B cells .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-DISEASE O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O

(C) Western blot analysis of HCC cancer stem cell markers in CRIP1-overexpressing Huh7 and Hep3B cells.


( H ) Tumor sphere formation assay in CRIP1 - knockdown MHCC - 97H and HepG2 cells . The data are shown as the relative fold change spheres compared with the siCtrl group ( right ) . Scale bar : 50 μm . Data information : Data represent the mean ± SD at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t - test H ,

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tumor sphere formation assay in CRIP1-knockdown MHCC-97H and HepG2 cells. The data are shown as the relative fold change spheres compared with the siCtrl group (right). Scale bar: 50 μm. Data information: Data represent the mean ± SD at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test H,


( I ) Western blot analysis of HCC cancer stem cell markers in CRIP1 - knockdown MHCC - 97H and HepG2 cells .

(I) Western blot analysis of HCC cancer stem cell markers in CRIP1-knockdown MHCC-97H and HepG2 cells.


( G ) Western blot analysis of cancer stem cell markers in the indicated groups .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O O O O O O

(G) Western blot analysis of cancer stem cell markers in the indicated groups.


( A ) Western blot analysis of BBOX1 protein levels in CRIP1 - overexpressing Huh7 and Hep3B cells . ( B ) Western blot analysis of BBOX1 protein levels in CRIP1 knockdown MHCC - 97H and HepG2 cells .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O

(A) Western blot analysis of BBOX1 protein levels in CRIP1-overexpressing Huh7 and Hep3B cells. (B) Western blot analysis of BBOX1 protein levels in CRIP1 knockdown MHCC-97H and HepG2 cells.


( D ) Western blot analysis of BBOX1 in CRIP1 - overexpressing Huh7 cells treated with CHX ( 100 μg / ml ) for the indicated durations ( lfte ) . The graph shows quantification of relative BBOX1 levels ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . * P < 0 . 05 and * * P < 0 . 01 . Differences were tested using an unpaired 2 - tailed Student ' s t - test D ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Western blot analysis of BBOX1 in CRIP1-overexpressing Huh7 cells treated with CHX (100 μg/ml) for the indicated durations (lfte). The graph shows quantification of relative BBOX1 levels (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01. Differences were tested using an unpaired 2-tailed Student's t-test D).


( E ) Western blot analysis of BBOX1 protein levels in CRIP1 - overexpressing Huh7 and Hep3B cells treated with MG132 ( 10 μM ) for 10 h . D ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O

(E) Western blot analysis of BBOX1 protein levels in CRIP1-overexpressing Huh7 and Hep3B cells treated with MG132 (10 μM) for 10 h. D).


( F ) Co - immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1 - overexpressing Huh7 and Hep3B cells .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O

(F) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1-overexpressing Huh7 and Hep3B cells.


( G ) Co - immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1 - overexpressing Huh7 and Hep3B cells transfected with STUB1 siRNAs .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O O

(G) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1-overexpressing Huh7 and Hep3B cells transfected with STUB1 siRNAs.


( H ) Co - immunoprecipitation and immunoblot analyses of the interaction among endogenous CRIP1 , STUB1 , and BBOX1 in Huh7 and Hep3B cells .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O B-CELL_LINE O O

(H) Co-immunoprecipitation and immunoblot analyses of the interaction among endogenous CRIP1, STUB1, and BBOX1 in Huh7 and Hep3B cells.


( I ) GST pulldown assay analysis of the association of CRIP1 with BBOX1 and STUB1 .

O O O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O

(I) GST pulldown assay analysis of the association of CRIP1 with BBOX1 and STUB1.


( J ) Co - immunoprecipitation and immunoblot analyses of the interaction between STUB1 and BBOX1 and BBOX1 ubiquitination in Huh7 cells transfected with STUB1 - Myc and CRIP1 siRNAs .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O

(J) Co-immunoprecipitation and immunoblot analyses of the interaction between STUB1 and BBOX1 and BBOX1 ubiquitination in Huh7 cells transfected with STUB1-Myc and CRIP1 siRNAs.


( B ) Co - immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in STUB1 - overexpressing Huh7 and Hep3B cells transfected with BBOX1 - WT or BBOX1 K240R mutant .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD O O O

(B) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant.


( C ) Western blot analysis of BBOX1 - WT or K240R mutant protein expression in Huh7 and Hep3B cells transfected with increasing amounts of STUB1 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-CELL_LINE O O O O O O B-GENEPROD O

(C) Western blot analysis of BBOX1-WT or K240R mutant protein expression in Huh7 and Hep3B cells transfected with increasing amounts of STUB1.


tumor sphere formation ability and quantification of spheres ( E ) , in STUB1 - overexpressing Huh7 and Hep3B cells transfected with BBOX1 - WT or BBOX1 K240R mutant . Scale bar : 50 μm . Data information : Data represent the mean ± SD of at least three independent experiments . * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t -

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

tumor sphere formation ability and quantification of spheres (E), in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant. Scale bar: 50 μm. Data information: Data represent the mean ± SD of at least three independent experiments. **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test


protein levels of HCC cancer stem cell markers ( G ) in STUB1 - overexpressing Huh7 and Hep3B cells transfected with BBOX1 - WT or BBOX1 K240R mutant .

B-EXP_ASSAY I-EXP_ASSAY O B-DISEASE O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD O O O

protein levels of HCC cancer stem cell markers (G) in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant.


Representative images of tumor sphere formation and quantification of spheres ( C ) in CRIP1 - overexpressing Huh7 cells with or without iCRT3 ( 50 μM ) for 24 h . Scale bar : 50 μm . Data information : Data represent the mean ± SD of at least three independent experiments . * * P < 0 . 01 and * * * P < 0 . 001 . Differences were tested using an unpaired 2 - tailed Student ' s t -

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of tumor sphere formation and quantification of spheres (C) in CRIP1-overexpressing Huh7 cells with or without iCRT3 (50 μM) for 24 h. Scale bar: 50 μm. Data information: Data represent the mean ± SD of at least three independent experiments. **P <0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test


( E ) Western blot analysis of HCC cancer stem cell markers in CRIP1 - overexpressing Huh7 cells with or without iCRT3 ( 50 μM ) for 24 h .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-DISEASE O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O

(E) Western blot analysis of HCC cancer stem cell markers in CRIP1-overexpressing Huh7 cells with or without iCRT3 (50 μM) for 24 h.


( F ) Coimmunoprecipitation and immunoblot analyses of β - catenin acetylation in CRIP1 knockdown MHCC - 97H cells treated with mildronate ( 5 mM ) for 24 h .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O

(F) Coimmunoprecipitation and immunoblot analyses of β-catenin acetylation in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24 h.


( G ) Western blot analysis of cytoplasmic and nuclear fractions of β - catenin in CRIP1 knockdown MHCC - 97H cells treated with mildronate ( 5 mM ) for 24h .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O

(G) Western blot analysis of cytoplasmic and nuclear fractions of β-catenin in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24h.


( I ) Co - immunoprecipitation and immunoblot analyses of β - catenin ubiquitination in CRIP1 knockdown MHCC - 97H cells treated with mildronate ( 5 mM ) and EX527 ( 20 μM ) for 24 h .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O

(I) Co-immunoprecipitation and immunoblot analyses of β-catenin ubiquitination in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) and EX527 (20 μM) for 24 h.


( A ) IHC analysis of CRIP1 , BBOX1 , β - catenin , CD133 , OCT4 , and NANOG using TAM ( left ) . Scale bars : 20 μm . Correlation analysis of CRIP1 expression with BBOX1 , β - catenin , CD133 , OCT4 , and NANOG expression in HCC tissues ( right ) . Data information : Data represent the mean ± SD of at least three independent experiments . . * * P < 0 . 01 and * * * P < 0 . 001 . Chi - square test was performed to determine the correlation ( A ) .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IHC analysis of CRIP1, BBOX1, β-catenin, CD133, OCT4, and NANOG using TAM (left). Scale bars: 20 μm. Correlation analysis of CRIP1 expression with BBOX1, β-catenin, CD133, OCT4, and NANOG expression in HCC tissues (right). Data information: Data represent the mean ± SD of at least three independent experiments.. **P <0.01 and ***P < 0.001. Chi-square test was performed to determine the correlation (A).


F : Full - length GST - tagged wild type ( wt ) and RG mutant p14ARF protein , GST alone as well as GST - PRMT1 were purified from bacteria , as visualized by SDS - PAGE and Coomassie Blue staining ( left and middle panel ) , with asterisks indicating the corresponding PRMT1 and p14ARF protein bands . GST and GST - p14ARF proteins ( wt , RG ) were subjected to in vitro - MT assays in the presence of GST - PRMT1 as described in ( B ) . Methylation activities were detected subsequent to protein blotting by autoradiography ( upper right panel ) . The asterisk highlights the methylated p14ARF wt protein , whereas the arrowhead indicates p14ARF - unrelated background signals ( likely deriving from PRMT1 automethylation ) . Amounts of p14ARF proteins were detected by immunostaining of the autoradiographed blot using α - p14ARF antibody ( lower right panel ) . Size markers ( in kDa ) are shown on the left .

O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

F: Full-length GST-tagged wild type (wt) and RG mutant p14ARF protein, GST alone as well as GST-PRMT1 were purified from bacteria, as visualized by SDS-PAGE and Coomassie Blue staining (left and middle panel), with asterisks indicating the corresponding PRMT1 and p14ARF protein bands. GST and GST-p14ARF proteins (wt, RG) were subjected to in vitro-MT assays in the presence of GST-PRMT1 as described in (B). Methylation activities were detected subsequent to protein blotting by autoradiography (upper right panel). The asterisk highlights the methylated p14ARF wt protein, whereas the arrowhead indicates p14ARF-unrelated background signals (likely deriving from PRMT1 automethylation). Amounts of p14ARF proteins were detected by immunostaining of the autoradiographed blot using α-p14ARF antibody (lower right panel). Size markers (in kDa) are shown on the left.


A , B : HeLa cells were transfected with empty vector ( e . v . , control ) or Myc - tagged wild type PRMT1 - containing plasmid . Immunofluorescence ( IF ) staining was performed using α - p14ARF ( red , endogenous p14ARF ) , α - Myc ( green , exogenous PRMT1 ) antibodies , DAPI ( blue , nuclei / DNA ) and Phalloidin ( grey , cytoplasm / F - actin ) . In ( A ) , representative IF results are shown , with asterisks indicating cells with exclusive nucleolar and arrowheads indicating cells with predominantly nucleo - / cytoplasmic p14ARF localization . The Merge displays the combination of p14ARF and PRMT1 staining . Scale bars : 15 μm . The quantification of exclusive nucleolar p14ARF was performed by cell counting ( percentage of cells ) and is shown for three independent experiments in ( B ) ( mean ± SD ) .

O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

A, B: HeLa cells were transfected with empty vector (e.v., control) or Myc-tagged wild type PRMT1-containing plasmid. Immunofluorescence (IF) staining was performed using α-p14ARF (red, endogenous p14ARF), α-Myc (green, exogenous PRMT1) antibodies, DAPI (blue, nuclei/DNA) and Phalloidin (grey, cytoplasm/F-actin). In (A), representative IF results are shown, with asterisks indicating cells with exclusive nucleolar and arrowheads indicating cells with predominantly nucleo-/cytoplasmic p14ARF localization. The Merge displays the combination of p14ARF and PRMT1 staining. Scale bars: 15 μm. The quantification of exclusive nucleolar p14ARF was performed by cell counting (percentage of cells) and is shown for three independent experiments in (B) (mean ± SD).


C , D : U2OS cells were transfected with EGFP - tagged p14ARF alone or in combination with Myc - tagged wild type PRMT1 - containing plasmids . p14ARF - EGFP ( green ) , DAPI ( blue , nuclei / DNA ) and PRMT1 ( red , using α - Myc antibody ) were visualized by fluorescence microscopy , for which representative results are shown in ( C ) , with the lower images displaying a magnification as indicated in the upper images by the rectangles . Scale bars : 10 μm . The subcellular distribution of p14ARF - EGFP ( exclusively nucleolar or not - exclusively nucleolar but predominantly nucleo - / cytoplasmic ) was quantified in p14ARF - positive and p14ARF / Myc - PRMT1 - double positive cells by cell counting ( percentage of cells ) for three independent experiments in ( D ) ( mean ± SD , * * * : p - value ≤ 0 . 001 using Welch ´ s t - test ) .

O O O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D: U2OS cells were transfected with EGFP-tagged p14ARF alone or in combination with Myc-tagged wild type PRMT1-containing plasmids. p14ARF-EGFP (green), DAPI (blue, nuclei/DNA) and PRMT1 (red, using α-Myc antibody) were visualized by fluorescence microscopy, for which representative results are shown in (C), with the lower images displaying a magnification as indicated in the upper images by the rectangles. Scale bars: 10 μm. The subcellular distribution of p14ARF-EGFP (exclusively nucleolar or not-exclusively nucleolar but predominantly nucleo-/cytoplasmic) was quantified in p14ARF-positive and p14ARF/Myc-PRMT1-double positive cells by cell counting (percentage of cells) for three independent experiments in (D) (mean ± SD, ***: p-value ≤ 0.001 using Welch´s t-test).


HeLa cells were transfected with the indicated siRNAs ( two control / non - targeting siRNAs and three PRMT1 - specific siRNAs ) . The cellular p14ARF distribution was determined by immunofluorescence staining ( IF ) using α - p14ARF antibody . Representative IF images for the siControl _ 1 and siPRMT1 _ 1 condition are shown in ( G ) ( endogenous p14ARF in red and the merge additionally with DAPI in blue for nuclei / DNA ) . Scale bars : 15 μm .

B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O O

HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and three PRMT1-specific siRNAs). The cellular p14ARF distribution was determined by immunofluorescence staining (IF) using α-p14ARF antibody. Representative IF images for the siControl_1 and siPRMT1_1 condition are shown in (G) (endogenous p14ARF in red and the merge additionally with DAPI in blue for nuclei/DNA). Scale bars: 15 μm.


A , U2OS cells were transfected with EGFP - tagged wild type ( wt ) or mutant ( RG , RF , RK ) p14ARF - containing plasmids . p14ARF - EGFP ( green ) , endogenous NPM as a nucleolar marker ( red , using α - NPM antibody ) , DAPI ( blue , nuclei / DNA ) and Phalloidin ( grey , cytoplasm / F - actin ) were visualized by fluorescence microscopy , for which representative results are shown in ( A ) . Scale bars : 10 μm .

O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

A, U2OS cells were transfected with EGFP-tagged wild type (wt) or mutant (RG, RF, RK) p14ARF-containing plasmids. p14ARF-EGFP (green), endogenous NPM as a nucleolar marker (red, using α-NPM antibody), DAPI (blue, nuclei/DNA) and Phalloidin (grey, cytoplasm/F-actin) were visualized by fluorescence microscopy, for which representative results are shown in (A). Scale bars: 10 μm.


D : HeLa cells were transfected with empty vector ( e . v . , control ) or Myc - tagged wild type PRMT1 - containing plasmid . Immunoprecipitation ( IP ) of endogenous p14ARF or NPM was performed from cell lysates using the corresponding antibodies or IgG as negative control . IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies . Staining results of the IP reactions derive from the same blot and exposure times with the white lines indicating where tracks were cut .

O O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

D: HeLa cells were transfected with empty vector (e.v., control) or Myc-tagged wild type PRMT1-containing plasmid. Immunoprecipitation (IP) of endogenous p14ARF or NPM was performed from cell lysates using the corresponding antibodies or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies. Staining results of the IP reactions derive from the same blot and exposure times with the white lines indicating where tracks were cut.


C : HeLa cells were transfected with the indicated siRNAs ( five control / non - targeting siRNAs and four PRMT1 - specific siRNAs ) . PRMT1 depletion and endogenous p14ARF protein levels were analyzed by immunoblotting using α - PRMT1 , α - p14ARF and α - β - TUBULIN ( loading control ) antibodies . The p14ARF signals were densitometrically quantified and normalized to the respective β - TUBULIN signal , as specified by the numbers below the blot , with the siControl _ 2 condition set to 1 .

O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

C: HeLa cells were transfected with the indicated siRNAs (five control/non-targeting siRNAs and four PRMT1-specific siRNAs). PRMT1 depletion and endogenous p14ARF protein levels were analyzed by immunoblotting using α-PRMT1, α-p14ARF and α-β-TUBULIN (loading control) antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blot, with the siControl_2 condition set to 1.


PRMT1 - proficient ( - Dox ) as well as PRMT1 - depleted cells ( + Dox ) were further treated with the protein synthesis inhibitor cycloheximide ( CHX ) and harvested after 0 , 0 . 5 , 1 , 2 , 3 , 4 , and 6 hours . Cell lysates were subjected to immunoblotting using α - p14ARF and α - β - TUBULIN ( loading control ) antibodies and conventional ECL detection ( F ) . For precise quantification of the p14ARF protein stability in the - / + Dox conditions during the CHX time course , immunoblotting was performed with independent cell lysates and primary antibodies , as in ( F ) , which were then detected using fluorescence dye - coupled secondary antibodies and the LI - COR Odyssey system . The quantitative p14ARF signals were normalized to the corresponding quantitative β - TUBULIN signals and displayed in ( G ) . The CHX - untreated samples ( 0 hour ) were set to 100 % for each condition .

B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O 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-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

PRMT1-proficient (-Dox) as well as PRMT1-depleted cells (+ Dox) were further treated with the protein synthesis inhibitor cycloheximide (CHX) and harvested after 0, 0.5, 1, 2, 3, 4, and 6 hours. Cell lysates were subjected to immunoblotting using α-p14ARF and α-β-TUBULIN (loading control) antibodies and conventional ECL detection (F). For precise quantification of the p14ARF protein stability in the -/+ Dox conditions during the CHX time course, immunoblotting was performed with independent cell lysates and primary antibodies, as in (F), which were then detected using fluorescence dye-coupled secondary antibodies and the LI-COR Odyssey system. The quantitative p14ARF signals were normalized to the corresponding quantitative β-TUBULIN signals and displayed in (G). The CHX-untreated samples (0 hour) were set to 100% for each condition.


HeLa cells were transfected with the indicated siRNAs ( two control / non - targeting siRNAs and two PRMT1 - specific siRNAs ) and irradiated at 150 J / cm2 UVC or not irradiated . After 24 hours , the cellular distribution of endogenous p14ARF was determined by immunofluorescence ( IF ) staining using α - p14ARF antibody ( red , endogenous p14ARF ) and in the merge additionally DAPI ( blue , nuclei / DNA ) . Representative IF results are shown for two conditions ( siControl _ 4 and siPRMT1 _ 1 ) in ( A ) , with asterisks indicating exclusively nucleolar and arrowheads indicating not - exclusively nucleolar but additionally or predominantly nucleo - / cytoplasmic p14ARF localization . Scale bars : 15 μm . The subcellular distribution of p14ARF , as determined by IF , was quantified by cell counting ( percentage of cells ) for all conditions ( B ) .

B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O 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-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY 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-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and two PRMT1-specific siRNAs) and irradiated at 150 J/cm2 UVC or not irradiated. After 24 hours, the cellular distribution of endogenous p14ARF was determined by immunofluorescence (IF) staining using α-p14ARF antibody (red, endogenous p14ARF) and in the merge additionally DAPI (blue, nuclei/DNA). Representative IF results are shown for two conditions (siControl_4 and siPRMT1_1) in (A), with asterisks indicating exclusively nucleolar and arrowheads indicating not-exclusively nucleolar but additionally or predominantly nucleo-/cytoplasmic p14ARF localization. Scale bars: 15 μm. The subcellular distribution of p14ARF, as determined by IF, was quantified by cell counting (percentage of cells) for all conditions (B).


H : CRISPR / Cas9 control ( CTR _ 1 and CTR _ 2 ) or PRMT1 - deleted ( KO _ 1 and KO _ 2 ) HeLa cell lines were irradiated at 150 J / cm2 UVC or not irradiated . After 4 hours , immunoprecipitations ( IP ) of endogenous p14ARF were performed from cell lysates using α - p14ARF antibody or IgG as negative control . IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies . Short and long exposure times are specified .

O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

H: CRISPR/Cas9 control (CTR_1 and CTR_2) or PRMT1-deleted (KO_1 and KO_2) HeLa cell lines were irradiated at 150 J/cm2 UVC or not irradiated. After 4 hours, immunoprecipitations (IP) of endogenous p14ARF were performed from cell lysates using α-p14ARF antibody or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies. Short and long exposure times are specified.


A : Kaplan - Meier plot was generated using the transcriptome data set of PDAC from TGCA and OncoLnc to visualize the survival of PDAC patients with low or high PRMT1 transcript levels and short - term ( < 500 days , left plot ) and long - term survival ( > 500 days , right plot ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-DISEASE O O O O O O O B-EXP_ASSAY O B-DISEASE B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

A: Kaplan-Meier plot was generated using the transcriptome data set of PDAC from TGCA and OncoLnc to visualize the survival of PDAC patients with low or high PRMT1 transcript levels and short-term (<500 days, left plot) and long-term survival (>500 days, right plot).


B : Representative immunohistochemistry ( IHC ) stainings of PRMT1 ( brown ) are shown for normal human pancreatic tissue and three PDAC specimens deriving from the cohort of 75 patients . Based on the immunostaining intensity and the percentage of stained tumor cells , all specimens were divided into three PRMT1 expression scores : not elevated , moderately elevated and highly elevated PRMT1 expression in tumor cells in comparison to normal pancreas . Right images are magnifications of the left images , as indicated by the black rectangles . Scale bars in the left images : 100 μm . Scale bars in the right images : 50 μm .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O B-DISEASE O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O O O O O O 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: Representative immunohistochemistry (IHC) stainings of PRMT1 (brown) are shown for normal human pancreatic tissue and three PDAC specimens deriving from the cohort of 75 patients. Based on the immunostaining intensity and the percentage of stained tumor cells, all specimens were divided into three PRMT1 expression scores: not elevated, moderately elevated and highly elevated PRMT1 expression in tumor cells in comparison to normal pancreas. Right images are magnifications of the left images, as indicated by the black rectangles. Scale bars in the left images: 100 μm. Scale bars in the right images: 50 μm.


E : Immunofluorescence ( IF ) stainings of the 75 PDAC specimens were performed using α - p14ARF ( red ) , α - CK8 / 18 ( green , staining ductal cells in normal pancreas and neoplastic cells in PDAC ) antibodies and DAPI ( blue , nuclei / DNA ) . Representative IF images are shown for normal human pancreatic tissue , two PDAC specimens displaying no p14ARF expression and three PDAC specimens displaying strong p14ARF expression in tumor cells . Left and corresponding right images show the same tissue section . Asterisks indicate nucleolar p14ARF localization in neoplastic cells of PDAC . The last image pair shows a magnification of the image pair above ( indicated by the rectangle ) . Scale bars : 35 μm .

O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O B-DISEASE O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-DISEASE O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE I-TISSUE O O B-DISEASE O O O B-GENEPROD B-EXP_ASSAY O O B-DISEASE O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O O O O B-DISEASE O O 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: Immunofluorescence (IF) stainings of the 75 PDAC specimens were performed using α-p14ARF (red), α-CK8/18 (green, staining ductal cells in normal pancreas and neoplastic cells in PDAC) antibodies and DAPI (blue, nuclei/DNA). Representative IF images are shown for normal human pancreatic tissue, two PDAC specimens displaying no p14ARF expression and three PDAC specimens displaying strong p14ARF expression in tumor cells. Left and corresponding right images show the same tissue section. Asterisks indicate nucleolar p14ARF localization in neoplastic cells of PDAC. The last image pair shows a magnification of the image pair above (indicated by the rectangle). Scale bars: 35 μm.