Subcellular distribution of 53BP1 DRiPs in HeLa that were left untreated or treated with MG132 ( 10 µM ) 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 , were treated with MG132 , VER or EeyI alone for 5 h . Scale bars : 20 µm . Quantitation of % of with 53BP1 foci . were treated as described in ( A ) divided in three categories , on number of 53BP1 foci / nucleus : 0 , 1 - 2 > 3 . Number of counted / condition : 1112 - 1537 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 01 .

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

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

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 . number of colonies surviving 10 days after plating is shown . Control or treated with MG132 ( 10 µM ) 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 , 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 .

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

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 S6 . U87MG were treated with SAR405 or PI103 for 2 h at indicated concentrations . effect of compounds on phosphorylated Akt ( p - Akt ) Ser473 Thr308 residues , total Akt on phosphorylated S6 ( Ser240 Ser244 residues ) total S6 was measured using western blotting . One representative experiment from at least two independent experiments is shown . Full - blots are shown in Supplementary Figure 6 .

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

(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 were treated with DMSO ( upper panels ) or with 10 μM SAR405 for 16 h ( lower panels ) . 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 were treated with indicated of Vps34 inhibitor for 24 h . effect on p62 biomarker was analyzed by western blotting . GAPDH was used as a loading control . results are representative of at least two independent experiments . Full - blots are shown in Supplementary Figure 14 .

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

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

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

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

(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 H1299 lines were cultured either in fed or starved conditions with EBSS 10 μM hydroxychloroquine ( HCQ ) , as indicated . were treated at same time with SAR405 at different concentrations . LC3 - II was measured by western blotting , GAPDH was used as a loading control . One representative experiment for three independent experiments is shown . Full - blots are shown in Supplementary Figure 18 . ( d ) Histograms show LC3 - II / GAPDH for each condition in two lines . Black gray histograms correspond to fed condition EBSS conditions , respectively . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

(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 ) needed to reach 40 % inhibition ( IC40 ) of proliferation is indicated . top histograms indicate IC40 of SAR405 as a single agent in combination with everolimus in three lines . bottom histograms indicate IC40 of everolimus as a single agent in combination with SAR405 . IC40 values for combination of two compounds were determined using rays with an effective fraction ∼ 0 . 5 , corresponding to compounds that are in equipotent proportion in mixture ( Supplementary Fig . 20 ) . absolute IC40 determination of both compounds is represented as 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 with increasing WRN concentrations cruciform consisting of random inverted repeats ( random - IR ) . Top , quantitation of cruciform unfolding . amount of linear from " No " lane was subtracted from other samples . Averages shown ; n = 3 technical replicates ; error bars , .

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

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 , WRN E84A ( used at 10 nM ) , carried out either in presence or absence of ATP , or with ATP - ƔS . Averages shown ; n ≥ 3 technical replicates ; error bars , .

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 random - IR cruciform structure was used as a substrate . H Quantitation of assays such as in ( G ) . amount of linear from " No " lane was subtracted from other samples . Averages shown ; n ≥ 3 technical replicates ; error bars , .

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

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

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 cruciform within negative supercoiled , producing two bands of expected ( 607 bp 2119 bp , indicated by green arrows , lane 2 ) . Simultaneous incubation of substrate with SspI EcoRI allows EcoRI cutting at TA repeats ( lane 5 ) , while extruded cruciform is refractory to EcoRI digestion ( lane 4 , * indicates scDNA ) .

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

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 was subjected to Cruciform extrusion procedure , where indicated . Supercoiled ( cruciform , refractory to EcoRI ) linear ( no cruciform , by EcoRI ) were resolved on a 1 % agarose gel , stained with GelRed . Top , quantitation of extruded cruciform in % with respect to amount of total that is present in lane . Averages shown ; n = 4 technical replicates ; error bars , . 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 . amount of linear from " No " lane was subtracted from other samples . Averages shown ; n = 3 technical replicates ; error bars , .

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

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 S . cerevisiae Sgs1 used in this study . gel was stained with Coomassie Brilliant blue .

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

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


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

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

B 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 WRN - 3A . Cruciform consisting of random inverted repeats was used . G Quantitation of assays such as in ( F ) . Averages shown ; n = 3 technical replicates ; error bars , .

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

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 - truncation WRN variants used in this study are shown . gel was stained with Coomassie Brilliant blue .

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

B 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 , .

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

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 ) MutLγ ( MLH1 - MLH3 ) . B MutS homologue heterodimers used in this study : MutSα ( MSH2 - MSH6 ) , MutSβ ( MSH2 - MSH3 ) MutSγ ( MSH4 - MSH5 ) .

O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR 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 MutS MutL homologue heterodimers , using an oligonucleotide - Holliday junction as a substrate . Red asterisk indicates position of radioactive label .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) MutSβ - 3K902A ( MSH3 with mutation K902A , ATPase - deficient ) . gel was stained with Coomassie Brilliant blue .

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

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

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

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

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

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 C320R to K63 - linked diubiquitin was measured by polarization using FlAsH tagged K63 - linked diubiquitin in which proximal ubiquitin was fluorescently - labeled . Error bars indicate s . d . are on three measurements per data point . One representative experiment of two is shown .

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

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

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

(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 26S proteasome , from expressing either USP14 wild - type , C114A , or C114R . One representative experiment of two is shown .

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

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 ) of ER - lumenal localization ( G ) were quantified . Data are average from six different tumors . N = 6 Bars error bars indicate

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) PFS ( I ) on CRIP1 expression in 108 HCC . Data information : Data represent mean ± SD of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Differences were tested using log - rank ( H - I ) .

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

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

(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 of HCC cancer markers in CRIP1 - overexpressing Huh7 Hep3B .

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

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


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

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

(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 of HCC cancer markers in CRIP1 - knockdown MHCC - 97H HepG2 .

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

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


( G ) Western blot of cancer markers in indicated groups .

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


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

O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O 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 of BBOX1 in CRIP1 - overexpressing Huh7 treated with CHX ( 100 μg / ml ) for indicated durations ( lfte ) . graph shows of relative BBOX1 ( right ) . Data information : Data represent mean ± SD of at least three independent experiments . * P < 0 . 05 * * P < 0 . 01 . Differences were tested using an unpaired 2 - Student ' s t - D ) .

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

(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 of BBOX1 in CRIP1 - overexpressing Huh7 Hep3B treated with MG132 ( 10 μM ) for 10 h . D ) .

O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O 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 immunoblot analyses of BBOX1 ubiquitination in CRIP1 - overexpressing Huh7 Hep3B .

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

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


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

O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR 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 immunoblot analyses of interaction among endogenous CRIP1 , STUB1 , BBOX1 in Huh7 Hep3B .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O 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 of association of CRIP1 with BBOX1 STUB1 .

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

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


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

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 immunoblot analyses of BBOX1 ubiquitination in STUB1 - overexpressing Huh7 Hep3B transfected with BBOX1 - WT or BBOX1 K240R mutant .

O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 of BBOX1 - WT or K240R mutant expression in Huh7 Hep3B transfected with increasing amounts of STUB1 .

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

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


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

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


of HCC cancer markers ( G ) in STUB1 - overexpressing Huh7 Hep3B transfected with BBOX1 - WT or BBOX1 K240R mutant .

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

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

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 of HCC cancer markers in CRIP1 - overexpressing Huh7 with or without iCRT3 ( 50 μM ) for 24 h .

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

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

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

(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 of fractions of β - catenin in CRIP1 knockdown MHCC - 97H treated with mildronate ( 5 mM ) for 24h .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O 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 immunoblot analyses of β - catenin ubiquitination in CRIP1 knockdown MHCC - 97H treated with mildronate ( 5 mM ) EX527 ( 20 μM ) for 24 h .

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

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

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

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

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

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

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

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

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

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 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 / ) Phalloidin ( grey , cytoplasm / F - actin ) were visualized by microscopy , for which representative results are shown in ( A ) . Scale bars : 10 μm .

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

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 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 lysates using corresponding antibodies or IgG as negative control . IP reactions input lysates were analyzed by immunoblotting using indicated antibodies . results of IP reactions derive from same blot exposure times with lines indicating where tracks were cut .

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

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

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

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

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

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

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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 transcriptome data set of PDAC from TGCA OncoLnc to visualize survival of PDAC with low or high PRMT1 transcript short - term ( < 500 days , left plot ) long - term survival ( > 500 days , right plot ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: 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 ( ) stainings of PRMT1 ( brown ) are shown for normal human pancreatic tissue three PDAC specimens deriving from cohort of 75 . on immunostaining of stained , specimens were divided into three PRMT1 expression scores : not elevated , moderately elevated highly elevated PRMT1 expression in in comparison to normal pancreas . Right are magnifications of left , as indicated by black rectangles . Scale bars in left : 100 μm . Scale bars in right : 50 μm .

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

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

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

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.


B : PaTu8988t were treated with 0 , 1 , 3 , 10 μM gemcitabine ( GEM ) . After 48 hours , lysates were analyzed by immunoblotting using indicated antibodies . p14ARF signals were densitometrically quantified normalized to respective GAPDH signal , as specified by numbers below blots , with untreated condition ( - GEM ) set to 1 . of PARP cleavage was also densitometrically quantified is indicated below blot . C : PaTu8988t were treated with 3 μM gemcitabine ( GEM ) or left untreated ( 0 hour ) . After 0 , 6 , 12 , 24 48 hours , methylation of endogenous p14ARF was analyzed by immunoblotting using α - me - p14ARF , α - p14ARF α - GAPDH ( loading control ) antibodies . methylated p14ARF p14ARF signals were densitometrically quantified normalized to respective GAPDH signal , as specified by numbers below blots , with untreated condition ( 0 hour ) set to 1 .

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

B: PaTu8988t cells were treated with 0, 1, 3, and 10 μM gemcitabine (GEM). After 48 hours, cell lysates were analyzed by immunoblotting using the indicated antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (- GEM) set to 1. The percentage of PARP cleavage was also densitometrically quantified and is indicated below the blot. C: PaTu8988t cells were treated with 3 μM gemcitabine (GEM) or left untreated (0 hour). After 0, 6, 12, 24 and 48 hours, methylation of endogenous p14ARF was analyzed by immunoblotting using α-me-p14ARF, α-p14ARF and α-GAPDH (loading control) antibodies. The methylated p14ARF and the p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (0 hour) set to 1.


D , PaTu8988t MiaPaCa - 2 were treated ( + ) with 20 μM MS023 or left untreated ( - ) for 3 days . For last 2 days , were additionally exposed to 0 , 1 , or 3 μM gemcitabine ( GEM ) . Subsequently , was quantified by flow cytometry ( in ( D ) ) using FITC - labeled AnnexinV propidium iodide ( PI ) for four independent experiments ( mean ± SD , * : p - value ≤ 0 . 05 using Welch ´ s t - ) .

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

D, PaTu8988t and MiaPaCa-2 cells were treated (+) with 20 μM MS023 or left untreated (-) for 3 days. For the last 2 days, the cells were additionally exposed to 0, 1, or 3 μM gemcitabine (GEM). Subsequently, cell death was quantified by flow cytometry (in (D)) using FITC-labeled AnnexinV and propidium iodide (PI) for four independent experiments (mean ± SD, *: p-value ≤ 0.05 using Welch´s t-test).


G : Apoptosis was quantified by flow cytometry ( G ) using FITC - labeled AnnexinV propidium iodide ( PI ) for six independent experiments ( mean ± SD , * * : p - value ≤ 0 . 005 , * : p - value ≤ 0 . 05 , ns : not significant using Welch ´ s t - ) .

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

G: Apoptosis was quantified by flow cytometry (G) using FITC-labeled AnnexinV and propidium iodide (PI) for six independent experiments (mean ± SD, **: p-value ≤ 0.005, *: p-value ≤ 0.05, ns: not significant using Welch´s t-test).


( A ) H1 - Hela were mock infected or infected with poliovirus ( MOI = 50 pfu / ) were lysed at indicated hours post - infection ( h . p . i . ) . Three independent experiments were measured for of p62 , blot shown is representative .

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

(A) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at the indicated hours post-infection (h.p.i.). Three independent experiments were measured for quantification of p62 levels, and the blot shown is representative.


( B ) H1 - Hela were mock infected or infected with poliovirus ( MOI = 50 pfu / ) were lysed at 6 h . p . i . Cycloheximide ( Cy . ) was added to mock infected at a final of 50 µg / mL , for 4 hours . were then pre - treated with 0 . 1 µM bafilomycin A1 ( Baf . A1 ) for 14 hours prior to infection kept under treatment throughout infection . MG132 was used at a final of 20 µM added to media at time of infection . blot shown is representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at 6 h.p.i. Cycloheximide (Cy.) was added to mock infected cells at a final concentration of 50 µg/mL, for 4 hours. Cells were then pre-treated with 0.1 µM bafilomycin A1 (Baf.A1) for 14 hours prior to infection and kept under treatment throughout infection. MG132 was used at a final concentration of 20 µM and added to the media at the time of infection. The blot shown is representative of three independent experiments.


( A ) were transfected with scrambled ( SCR ) or anti - LC3 ( LC3 ) siRNAs , 48 h later infected with PV at an MOI of 50 pfu / , or mock infected . were collected at 6 h . p . i . , immunoblots were performed on lysates for p62 , GAPDH , LC3 .

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

(A) Cells were transfected with scrambled (SCR) or anti-LC3 (LC3) siRNAs, and 48 h later infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i., and immunoblots were performed on lysates for p62, GAPDH, and LC3.


( B ) were treated with 10 mM 3 - MA for 12 h , then infected with PV at an MOI of 50 pfu / , or mock infected . were collected at 6 h . p . i . lysates were used for immunoblots .

O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were treated with 10 mM 3-MA for 12 h, then infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i. and lysates were used for immunoblots.


Triplicate samples of H1 - Hela were infected with PV at an MOI of 0 . 1 pfu / ( A ) or MOI of 50 pfu / ( B ) . were pre - treated with 20 µM leupeptin for 14 hours prior to infection kept under treatment throughout infection . - associated was collected at indicated times post - infection , titers were determined by plaque . Parallel infections were collected for Western blots of LC3 ( A ) or p62 ( B ) In ( B ) - associated was collected at 6 h . p . i . for plaque .

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

Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell (A) or MOI of 50 pfu/cell (B). Cells were pre-treated with 20 µM leupeptin for 14 hours prior to infection and kept under treatment throughout infection. Cell-associated virus was collected at the indicated times post-infection, and virus titers were determined by plaque assay. Parallel infections were collected for Western blots of LC3 (A) or p62 (B) In (B) cell-associated virus was collected at 6 h.p.i. for plaque assay.


( C ) Infections were performed as in ( A ) , then 10 µg / mL each of E64d Pepstatin A ( Pep . A ) were added to media at time of infection . Inhibition of lysosomal degradation was confirmed by immunoblot for LC3 GAPDH . ( D ) Infection at an MOI of 50 pfu / , with E64 / Pep . A as in ( C ) .

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

(C) Infections were performed as in (A), then 10 µg/mL each of E64d and Pepstatin A (Pep.A) were added to the media at the time of infection. Inhibition of lysosomal degradation was confirmed by immunoblot for LC3 and GAPDH. (D) Infection at an MOI of 50 pfu/cell, with E64/Pep.A as in (C).


( A ) Triplicate samples of H1 - Hela were infected with PV at an MOI of 0 . 1 pfu / , - associated was collected at indicated times post - infection . NH4Cl ( 20 mM ) was added to media at time of infection ( solid ) . titers were then determined by plaque . ( B ) Infection as in ( A ) , carried out to 16 h to represent a multiple - cycle infection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, and cell-associated virus was collected at the indicated times post-infection. NH4Cl (20 mM) was added to the media at the time of infection (solid line). Virus titers were then determined by plaque assay. (B) Infection as in (A), carried out to 16 h to represent a multiple-cycle infection.


( C ) Triplicate H1 - Hela infections with PV at an MOI of 50 pfu / , with - associated collected at 6 h . p . i . ( D ) were infected as in ( A ) , then NH4Cl was added to half of samples at 3 . 5 hours post - infection , - associated was collected immediately or at 7 h . p . i .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Triplicate H1-Hela infections with PV at an MOI of 50 pfu/cell, with cell-associated virus collected at 6 h.p.i. (D) Cells were infected as in (A), then NH4Cl was added to half of the samples at 3.5 hours post-infection, and cell-associated virus was collected immediately or at 7 h.p.i.


( E ) were pre - treated for 14 hours with 0 . 1 µM bafilomycin A1 ( Baf . A1 ) kept under treatment throughout infection ( MOI = 0 . 1 pfu / ) . - associated was collected at 6 or 12 h . p . i . titers were determined by plaque . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 0001 .

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

(E) Cells were pre-treated for 14 hours with 0.1 µM bafilomycin A1 (Baf.A1) and kept under treatment throughout infection (MOI = 0.1 pfu/cell). Cell-associated virus was collected at 6 or 12 h.p.i. and virus titers were determined by plaque assay. * p<0.05, ** p<0.01, *** p<0.0001.


( A ) H1 - HeLa were infected with PV at an MOI of 50 pfu / . were pulsed at indicated h . p . i with 35S - labeled methionine for 1 h then lysed . Lysates were run on SDS - PAGE . Expected viral are labeled according to recognized banding patterns .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H1-HeLa cells were infected with PV at an MOI of 50 pfu/cell. Cells were pulsed at the indicated h.p.i with 35S-labeled methionine for 1 h then lysed. Lysates were run on SDS-PAGE. Expected viral proteins are labeled according to recognized banding patterns.


( B ) Triplicate plates of H1 - Hela were infected with PV at an MOI of 0 . 1 pfu / , host GAPDH were measured by qRT - PCR . were normalized to GAPDH using delta - Ct method . NH4Cl treatment was as described in Figure 4 , Guanidine HCl ( 2 mM ) was added to media at time of infection . data shown are pooled from three replicate experiments , titer of - associated collected at 6h . p . i . from each replicate was determined by plaque .

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

(B) Triplicate plates of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, virus RNA and host GAPDH RNA were measured by qRT-PCR. Virus RNA levels were normalized to GAPDH levels using the delta-Ct method. NH4Cl treatment was as described in Figure 4, and Guanidine HCl (2 mM) was added to the media at the time of infection. The data shown are pooled from three replicate experiments, and the titer of cell-associated virus collected at 6h.p.i. from each replicate was determined by plaque assay.


( C ) Triplicate plates of 293T were treated with 20 mM 3 - MA for 2 hours prior to infection , kept under treatment throughout infection . PV infections were done at an MOI of 0 . 1 pfu / . titers were analyzed as in ( B ) . * * p < 0 . 01 , * * * p < 0 . 0001 .

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

(C) Triplicate plates of 293T cells were treated with 20 mM 3-MA for 2 hours prior to infection, and kept under treatment throughout infection. PV infections were done at an MOI of 0.1 pfu/cell. RNA levels and virus titers were analyzed as in (B). ** p<0.01, *** p<0.0001.


( A ) H1 - Hela were infected at an MOI of 50 pfu / , half of samples were treated with NH4Cl . were labeled with 35S - Methionine from 3 h . p . i . until collection at 5 or 6 h . p . i . , lysates were then separated on a 15 - 30 % sucrose gradients . Fractions were then collected per minute ( CPM ) were analyzed for each fraction . Representative gradients from three independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with NH4Cl. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 or 6 h.p.i., and lysates were then separated on a 15-30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction. Representative gradients from three independent experiments are shown.


. ( B ) three fractions representing 150S peak in each experiment were pooled for plaque . Data shown are averages from 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) The three fractions representing the 150S peak in each experiment were pooled for plaque assay analysis. Data shown are the averages from three independent experiments. (


( C ) Three fractions representing 150S 75S peaks were pooled run on SDS - PAGE , 35S - Methionine labeled bands were visualized . bands are labeled according to expected relative migration pattern , VP2 is identified by its absence in 75S peak . of VP0 to VP3 bands was analyzed from four independent experiments plotted in arbitrary units . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 0001 .

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

(C) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE, and the 35S-Methionine labeled bands were visualized. The bands are labeled according to expected relative migration pattern, and VP2 is identified by its absence in the 75S peak. The ratio of VP0 to VP3 bands was analyzed from four independent experiments and plotted in arbitrary units. * p<0.05, ** p<0.01, *** p<0.0001.


( A ) H1 - Hela were infected at an MOI of 50 pfu / , half of samples were treated with bafilomycin A1 as described in Figure 4 . were labeled with 35S - Methionine from 3 h . p . i . until collection at 5 h . p . i . , lysates were then separated on a 15 - 30 % sucrose gradients . Fractions were then collected per minute ( CPM ) were analyzed for each fraction .

(A) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with bafilomycin A1 as described in Figure 4. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 h.p.i., and lysates were then separated on a 15-30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction.


( B ) Three fractions representing 150S 75S peaks were pooled run on SDS - PAGE as in Figure 6 . of VP0 to VP3 bands was analyzed plotted in arbitrary units . titer of infectious in pooled 150S fractions was determined by plaque . * p < 0 . 05 .

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

(B) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE as in Figure 6. The ratio of VP0 to VP3 bands was analyzed and plotted in arbitrary units. The titer of the infectious virus in the pooled 150S fractions was determined by plaque assay. * p<0.05.


D . model consist of a fusion between Ura3p GFP ( URA3sol , left ) amyloid - β - peptide of 42 residues ( Aβ ) ( URA3agg , right ) . After 18 hours of expression Ura3psol remains homogeneously distributed whereas Ura3pagg is accumulated into intracellular foci .

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

D. The model proteins consist of a fusion between Ura3p and GFP (URA3sol, left) and the amyloid-β-peptide of 42 residues (Aβ) (URA3agg, right). After 18 hours of expression Ura3psol remains homogeneously distributed whereas Ura3pagg is accumulated into intracellular foci.