EMBO/SourceData huggingface dataset:
Figure 5Immunoprecipitation of p53 and AGR2 in the digitonin fraction of A549 cells. A549 cells were treated with Tunicamycin (Tm 100ng/ml), Thapsigargin (Tg 25nM) and Brefeldin A (BFA 0,1 and 0,25nM). Endogenous p53 was immunoprecipitated from the cytoplasmic fraction (digitonin fraction) of A549 cells. Coprecipitated endogenous AGR2 was detected by western blot.A549 were treated with Etoposide (Eto) for 2 hours to induce p53 pathway. Then cells have been challenged with Tm, Tg and BFA at the indicated concentrations for 16 hours. Luciferase experiments were performed after 24 hours of transfection. Graph shows the fold induction of p53 luciferase construct. Western blot experiments for phospho-p53, pan-p53, p21 and HSP90 were performed as control. n=3, biological replicates ﻿(mean ± SD; ﻿Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated. *p<0.05, ***p < 0.001.A549 were transfected with control siRNA (siC) and AGR2-tergetd siRNA (siAGR2). After 24 hours, cells were transfected with p53-luciferase construct. Cells were then treated as in (C) and luciferase experiments were performed. n=4, biological replicates ﻿(mean ± SD. ﻿Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated.Left: Western blot experiments for phospho-p53, pan-p53, and p21 in AGR2-silenced A549 cells exposed to ER stressors: 100ng/ml Tunicamycin ( Tm), 25nM Thapsigargin (Tg) and 0.25 nM Brefeldin A (BFA), in the presence and absence of etoposide. Right: Quantification of the western blot. n=3, biological replicates, mean ±SD.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.
Figure 1(A) Western blot analysis of CRIP1 protein expression in 16 paired HCC tumor tissues (T) and adjacent nontumor tissues (N). The graph represents CRIP1 protein expression normalized to GAPDH levels (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, paired (A, Student's t-test(B) Representative IHC images of CRIP1 protein expression in 108 paired HCC tumor tissues and adjacent nontumor tissues. Scale bars: 20 μm. Statistical analysis of immunohistochemical score of CRIP1 expression in HCC tumor tissues and paired adjacent tissues (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, paired B) Student's t-test(C) CRIP1 mRNA expression in the TCGA-LIHC dataset. (D) mRNA levels of CRIP1 in the GSE36376 dataset from the GEO database. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, unpaired Student's t-testThe protein (F) levels of CRIP1 in HCC cell lines and normal LO2 hepatocytes. (G) Relative CRIP1 protein level compared to normal LO2 hepatocytes. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, unpaired G) Student's t-test(H-I) Kaplan-Meier survival analyses of OS (H) and PFS (I) based on CRIP1 expression levels in 108 HCC patients. Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using log-rank test (H-I).
Figure 2(B) Tumor sphere formation assay in CRIP1-overexpressing Huh7 and Hep3B cells. The data are shown as the relative fold change spheres compared with the vector group (right). Scale bar: 50 μm. Data information: Data represent the mean ± SD at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test (B,(C) Western blot analysis of HCC cancer stem cell markers in CRIP1-overexpressing Huh7 and Hep3B cells.(H) Tumor sphere formation assay in CRIP1-knockdown MHCC-97H and HepG2 cells. The data are shown as the relative fold change spheres compared with the siCtrl group (right). Scale bar: 50 μm. Data information: Data represent the mean ± SD at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test H,(I) Western blot analysis of HCC cancer stem cell markers in CRIP1-knockdown MHCC-97H and HepG2 cells.
Figure 4(A) Western blot analysis of BBOX1 protein levels in CRIP1-overexpressing Huh7 and Hep3B cells. (B) Western blot analysis of BBOX1 protein levels in CRIP1 knockdown MHCC-97H and HepG2 cells.(D) Western blot analysis of BBOX1 in CRIP1-overexpressing Huh7 cells treated with CHX (100 μg/ml) for the indicated durations (lfte). The graph shows quantification of relative BBOX1 levels (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01. Differences were tested using an unpaired 2-tailed Student's t-test D).(E) Western blot analysis of BBOX1 protein levels in CRIP1-overexpressing Huh7 and Hep3B cells treated with MG132 (10 μM) for 10 h. D).(F) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1-overexpressing Huh7 and Hep3B cells.(G) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1-overexpressing Huh7 and Hep3B cells transfected with STUB1 siRNAs.(H) Co-immunoprecipitation and immunoblot analyses of the interaction among endogenous CRIP1, STUB1, and BBOX1 in Huh7 and Hep3B cells.(I) GST pulldown assay analysis of the association of CRIP1 with BBOX1 and STUB1.(J) Co-immunoprecipitation and immunoblot analyses of the interaction between STUB1 and BBOX1 and BBOX1 ubiquitination in Huh7 cells transfected with STUB1-Myc and CRIP1 siRNAs.
Figure 5(B) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant.(C) Western blot analysis of BBOX1-WT or K240R mutant protein expression in Huh7 and Hep3B cells transfected with increasing amounts of STUB1.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-testprotein levels of HCC cancer stem cell markers (G) in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant.
Figure 6Representative images of tumor sphere formation and quantification of spheres (C) in CRIP1-overexpressing Huh7 cells with or without iCRT3 (50 μM) for 24 h. Scale bar: 50 μm. Data information: Data represent the mean ± SD of at least three independent experiments. **P <0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test(E) Western blot analysis of HCC cancer stem cell markers in CRIP1-overexpressing Huh7 cells with or without iCRT3 (50 μM) for 24 h.(F) Coimmunoprecipitation and immunoblot analyses of β-catenin acetylation in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24 h.(G) Western blot analysis of cytoplasmic and nuclear fractions of β-catenin in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24h.(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.
Figure 7(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).
Figure 1F: 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.
Figure 2A, B: HeLa cells were transfected with empty vector (e.v., control) or Myc-tagged wild type PRMT1-containing plasmid. Immunofluorescence (IF) staining was performed using α-p14ARF (red, endogenous p14ARF), α-Myc (green, exogenous PRMT1) antibodies, DAPI (blue, nuclei/DNA) and Phalloidin (grey, cytoplasm/F-actin). In (A), representative IF results are shown, with asterisks indicating cells with exclusive nucleolar and arrowheads indicating cells with predominantly nucleo-/cytoplasmic p14ARF localization. The Merge displays the combination of p14ARF and PRMT1 staining. Scale bars: 15 μm. The quantification of exclusive nucleolar p14ARF was performed by cell counting (percentage of cells) and is shown for three independent experiments in (B) (mean ± SD).C, D: U2OS cells were transfected with EGFP-tagged p14ARF alone or in combination with Myc-tagged wild type PRMT1-containing plasmids. p14ARF-EGFP (green), DAPI (blue, nuclei/DNA) and PRMT1 (red, using α-Myc antibody) were visualized by fluorescence microscopy, for which representative results are shown in (C), with the lower images displaying a magnification as indicated in the upper images by the rectangles. Scale bars: 10 μm. The subcellular distribution of p14ARF-EGFP (exclusively nucleolar or not-exclusively nucleolar but predominantly nucleo-/cytoplasmic) was quantified in p14ARF-positive and p14ARF/Myc-PRMT1-double positive cells by cell counting (percentage of cells) for three independent experiments in (D) (mean ± SD, ***: p-value ≤ 0.001 using Welch´s t-test).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.
Figure 3A, U2OS cells were transfected with EGFP-tagged wild type (wt) or mutant (RG, RF, RK) p14ARF-containing plasmids. p14ARF-EGFP (green), endogenous NPM as a nucleolar marker (red, using α-NPM antibody), DAPI (blue, nuclei/DNA) and Phalloidin (grey, cytoplasm/F-actin) were visualized by fluorescence microscopy, for which representative results are shown in (A). Scale bars: 10 μm.D: HeLa cells were transfected with empty vector (e.v., control) or Myc-tagged wild type PRMT1-containing plasmid. Immunoprecipitation (IP) of endogenous p14ARF or NPM was performed from cell lysates using the corresponding antibodies or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies. Staining results of the IP reactions derive from the same blot and exposure times with the white lines indicating where tracks were cut.
Figure 4C: HeLa cells were transfected with the indicated siRNAs (five control/non-targeting siRNAs and four PRMT1-specific siRNAs). PRMT1 depletion and endogenous p14ARF protein levels were analyzed by immunoblotting using α-PRMT1, α-p14ARF and α-β-TUBULIN (loading control) antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blot, with the siControl_2 condition set to 1.PRMT1-proficient (-Dox) as well as PRMT1-depleted cells (+ Dox) were further treated with the protein synthesis inhibitor cycloheximide (CHX) and harvested after 0, 0.5, 1, 2, 3, 4, and 6 hours. Cell lysates were subjected to immunoblotting using α-p14ARF and α-β-TUBULIN (loading control) antibodies and conventional ECL detection (F). For precise quantification of the p14ARF protein stability in the -/+ Dox conditions during the CHX time course, immunoblotting was performed with independent cell lysates and primary antibodies, as in (F), which were then detected using fluorescence dye-coupled secondary antibodies and the LI-COR Odyssey system. The quantitative p14ARF signals were normalized to the corresponding quantitative β-TUBULIN signals and displayed in (G). The CHX-untreated samples (0 hour) were set to 100% for each condition.
Figure 5HeLa 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).
Figure 6H: 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.
Figure 7A: Kaplan-Meier plot was generated using the transcriptome data set of PDAC from TGCA and OncoLnc to visualize the survival of PDAC patients with low or high PRMT1 transcript levels and short-term (<500 days, left plot) and long-term survival (>500 days, right plot).B: Representative immunohistochemistry (IHC) stainings of PRMT1 (brown) are shown for normal human pancreatic tissue and three PDAC specimens deriving from the cohort of 75 patients. Based on the immunostaining intensity and the percentage of stained tumor cells, all specimens were divided into three PRMT1 expression scores: not elevated, moderately elevated and highly elevated PRMT1 expression in tumor cells in comparison to normal pancreas. Right images are magnifications of the left images, as indicated by the black rectangles. Scale bars in the left images: 100 μm. Scale bars in the right images: 50 μm.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.
Figure 8B: 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 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 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).
Figure 1(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 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.
Figure 2(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) 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.
Figure 3Triplicate 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 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).
Figure 4(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/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) 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.
Figure 5(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 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 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.
Figure 6(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) 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 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.
Figure 7(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 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.
Figure 1D. 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.