Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Dotplot of Wilcoxon rank-sum test p-values for PRMT5 and MEP50 expression in cancers with data collected from the TCGA. Upregulated expression (purple) and downregulated (green) are shown in dots scaled by–log( p ). Corresponding cancer cell lines probed in this study are bolded. B. Heatmap of gene expression of PRMT5 and MEP50 from TCGA lung cancer RNA-Seq data in normal and patients with lung adenocarcinoma (LUAD) or squamous cell carcinoma (LUSC), ranked by sample type (tumor or normal tissue) and then Shedden poor survival markers showing substantially increased expression of PRMT5 and MEP50 relative to normal tissue and poor survival markers. C. Dot-plot showing distribution of TCGA individual sample data for LUAD or LUSC; mean FPKM expression RNA-Seq value shown by black bar; Comparison between tumor and normal tissue expression p < 0.001 from Mann-Whitney Rank Sum Test. D. Whole cell lysate immunoblots for PRMTs and MEP50 in the normal and corresponding cancer cell lines. GAPDH is a loading control. E. Whole cell lysate and chromatin extracted from IMR90 and A549 probed with indicated antibodies. GAPDH and H3 are controls for lysate and chromatin immunoblots, respectively. F. Immunoblots for PRMT5 or MEP50 from A549 cells expressing shRNA targeted against GFP as a control (GFP kd ), against PRMT5 (PRMT5 kd ) or against MEP50 (MEP50 kd ). G. Representative immunofluorescence images for PRMT5 (red) or MEP50 (red) and counterstained with DAPI (blue) from GFP kd , PRMT5 kd , and MEP50 kd A549 cells demonstrating primarily cytoplasmic localization. Scale bar, 50 μm. H. Blue-native gel immunoblots for PRMT5 or MEP50 from knockdown A549 cells as in E. Native Molecular mass markers are indicated. Recombinant human PRMT5-MEP50 complex ( Hs PRMT5-MEP50) or SDS/boiled Hs PRMT5-MEP50 with SDS (lanes 1 and 2) are used for identification of intact complex (indicated) or free PRMT5 and MEP50, respectively.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Expressing, RNA Sequencing Assay, MANN-WHITNEY, Western Blot, shRNA, Immunofluorescence, Recombinant

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. FPKM ( F ragments p er k ilobase of transcript per m illion mapped reads) of all 9 PRMTs (CARM1 is PRMT4) and MEP50 from the 3 RNA-Seq replicates for each of the control (GFP kd ) and PRMT5 kd and MEP50 kd are shown on an arbitrary scale for each. PRMT5 and MEP50 reduced expression in each knockdown is indicated with the corresponding fold change from DESeq2 analysis. B. Top: piechart showing up- (purple) and down- (green) regulated genes as calculated by both DESeq2 and edgeR algorithms with cutoffs as indicated. Bottom: Venn diagram showing the overlap in the total geneset of altered expression between PRMT5 kd and MEP50 kd . C. Heatmap of the 955 genes altered in both knockdowns, showing PRMT5 and MEP50 up- (purple) and down- (green) regulated genes. The calculated Pearson correlation between the two knockdowns is shown. D. Selected ConsensusPathDB output genes in protein complexes are shown, colored by up- (purple) and down- (green) regulation. Bolded genes are viewed in E. E. IGV genome browser view of the RNA-Seq profile from a single replicate of the GFP kd , PRMT5 kd, and MEP50 kd analysis. The fibrinogen locus (chr4: FGB, FGA, and FGG) is shown as an example of downregulation upon knockdown (left panel, shaded green) and collagen 5α (chr9: COL5A1) is shown as an example of upregulation upon knockdown (right panel, shaded purple). F. Example gene set enrichment analysis of the 955 genes altered in both knockdowns. Top panel: adhesion molecules known to be downregulated in lung cancer had a significant positive normalized enrichment score (NES) upon knockdown. Bottom panel: a gene set known to be upregulated in lung and breast cancers had a significant negative NES upon knockdown. G. Dotplot of gene ontology terms enriched on both PRMT5 and MEP50 knockdown upregulated genes (top panel, purple) and downregulated genes (bottom panel, green) are shown in dots scaled by–log( p ). H. Dotplot of Ingenuity Pathway Analysis (IPA) diseases and functions enriched in both knockdowns are shown in dots scaled by–log( p ). I. Dotplot of Ingenuity Pathway Analysis (IPA) upstream pathways enriched in both knockdowns are shown in dots scaled by–log( p ).

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: RNA Sequencing Assay, Expressing

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Proliferation of A549 cells expressing shRNA targeted against GFP (red circle; GFP kd ), PRMT5 (blue square; PRMT5 kd ) and MEP50 (orange triangle; MEP50 kd ). Plotted data are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. Histogram (right): cell count on 6 th day for each sample. B. Colony formation assays of A549 cells expressing shRNAs are quantified for relative colony numbers (% of control). +Values are means ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. C. Migration through 8 μm pores by GFP kd , PRMT5 kd or MEP50 kd A549 cells was measured. Left: representative crystal violet staining of migrated cells on the underside of the porous polycarbonate membrane under a phase-contrast microscope (20X). Right: quantification of the migrated cells. Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. D. Matrigel invasion through 8 μm pores by GFP kd , PRMT5 kd or MEP50 kd A549 cells was measured. Left: representative crystal violet staining of invaded cells on the underside of the porous polycarbonate membrane under a phase-contrast microscope (20X). Right: quantification of invaded cells. Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. E. Rate of wound-healing of GFP kd , PRMT5 kd or MEP50 kd A549 cells was measured over 24 hours. Top: phase-contrast pictures (10X) of each cell line after scratching the confluent cells with the leading edge of cells indicated by a dashed yellow line. Bottom: quantification of the wound width (% 0hr wound width) after the scratch in indicated time points. Values are means ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. F. 3D spheroid cell invasion assay of GFP kd , PRMT5 kd or MEP50 kd A549 cells. Cells were aggregated into spheroids and then induced to invade the invasion matrix for the indicated time courses. The flat area of the cell mass view was calculated at four time points to measure cell invasion rate. Top: representative spheroid images for individual A549 cell lines under a phase-contrast microscope (20X). Bottom: histogram depicting the Spheroid area (×10 6 μm 2 ) measured with ImageJ 1.49. Values are means ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. G. A549 cells were treated with 0, 10nM, 100nM, 500nM, 1μM, or 5μM GS591 for 4 days and lysates or extracted histones were blotted for PRMT5, SYM10 (methylated SmD3), GAPDH (control), H4R3me2s, and H3 as indicated. DB71 stain of extracted histones is also shown. H. Invasivity of A549 cells treated with 500nM GSK591 for 0 or 4 days were measured in a Matrigel assay. Top: Representative crystal violet staining of invaded cells on the underside of the porous polycarbonate membrane through a phase-contrast microscope (20X) are shown. Bottom: Quantification of the invaded cells. Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. I. Morphology (top micrographs) and viability (bottom plot) of A549 or IMR90 cells treated with 5 μM GSK591 for 4 days. Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Expressing, shRNA, Cell Counting, Migration, Staining, Microscopy, Invasion Assay, Methylation, Matrigel Assay

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Representative cell morphology images of GFP kd , PRMT5 kd or MEP50 kd A549 cells. Cells were treated with (+) or without (−) 10 nM TGFβ1 for 2 days. Inserts show higher magnification views of selected cells. B. Invasivity of GFP kd , PRMT5 kd or MEP50 kd A549 cells treated with (+) or without (−) 10 nM TGFβ1 for 2 days were measured in a Matrigel assay. Left: Representative crystal violet staining of invaded cells on the underside of the porous polycarbonate membrane through a phase-contrast microscope (20X) are shown. Right: Quantification of the invaded cells (+TGFβ, blue bars). Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. C. Top: Images of a 3D spheroid cell invasion assay of GFP kd , PRMT5 kd or MEP50 kd A549 cells treated with (+) or without (−) 10 nM TGFβ1 for 2 days are shown. Bottom: Quantification of spheroid area (x10 6 μm 2 ) measured with ImageJ 1.49 (+TGFβ, blue bars). Values are means ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. D. Representative cell morphology images of A549 cells treated with (+) or without (−) 500 nM GSK591 for 4 days and (+) or without (−) 10 nM TGFβ1 for 2 days. Inserts show higher magnification views of selected cells. E. Invasivity of A549 lung adenocarcinoma cells, F. SK-MES-1 lung squamous cell carcinoma cells, and G. HS578T breast carcinoma cells treated with (+) or without (−) 500 nM GSK591 for 4 days and (+) or without (−) 10 nM TGFβ1 for 2 days measured in a Matrigel assay. Top: Representative crystal violet staining of invaded cells on the underside of the porous polycarbonate membrane through a phase-contrast microscope (20X) are shown. Bottom: Quantification of the invaded cells (+TGFβ, blue bars). Values are mean ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Matrigel Assay, Staining, Microscopy, Invasion Assay

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Immunoblots for PRMTs, EMT markers, spliceosome factors, histone methylarginine PTMs, and controls as indicated, from A549 cells expressing shRNA targeted against GFP as a control (GFP kd ), against PRMT5 (PRMT5 kd ) or against MEP50 (MEP50 kd ). These cells were treated with (+) or without (−) 10 nM of TGFβ1 for 2 days. B. Immunoblots for the indicated proteins from A549 cells treated with (+) or without (−) 10 nM of TGFβ1 for 2 days and treated with (+) or without (−) 500 nM GSK591 for 4 days. C. Immunoblots for PRMT5, MEP50, and histone methylarginine PTMs from H460 lung cancer cells and D. HS578T breast cancer cells treated with (+) or without (−) 10 nM of TGFβ1 for 2 days and treated with (+) or without (−) 500 nM GSK591 for 4 days. E. qRT-PCR of relative mRNA expression of A549 cells treated with (+, blue bars) or without (−) 10 nM TGFβ1 for 2 days.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Western Blot, Expressing, shRNA, Quantitative RT-PCR

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Relative mRNA levels of indicated genes in A549 cells (control, PRMT5 kd and MEP50 kd , as indicated on top) treated with 10 nM of TGFβ1 (blue bars) for 2 days were determined by qRT-PCR. β-Actin was used as an internal control. Values are means ± S.E.M. of three independent experiments. * p < 0.05 from two-tailed one-way ANOVA test. B. qRT-PCR for the indicated genes from A549 cells expressing shRNA targeted against GFP as a control (GFP kd ), against PRMT5 (PRMT5 kd ) or against MEP50 (MEP50 kd ). These cells were treated with (+, blue bars) or without (−) 10 nM of TGFβ1 for 2 days as indicated. Values are means ± S.E.M. of three independent experiments. * p < 0.05 from one-way ANOVA test. C. Heatmap generated by ChIP-qPCR values to demonstrate the histone methylarginine code surrounding the candidate genes measured in (A). ChIP-qPCR enrichments from H3R2me1, H3R2me2s, H3R8me2s, H4R3me1, H4R3me2a, H4R3me2s and histone H3 from A549 on primers −1kb, at the promoter, or +1kb of the indicated genes are arrayed from blue (no enrichment) to yellow (maximal enrichment). ChIP-qPCR of TGFβ1-downregulated genes are arrayed on the top and TGFβ1-upregulated genes are on the bottom. D. Selected ChIP-qPCR experiments of alternatively up-regulated (Snail1 and Vim) and down-regulated genes (CDH1 and SPDEF) from A549 cells (control, PRMT5 kd and MEP50 kd , as indicated on bottom) treated with 10 nM of TGFβ1 (blue bars) for 2 days are shown. The horizontal dotted line indicates the upper limit of the 95% confidence interval of the signal from no-antibody (NA) control ChIPs. Error bars, SEM (n=3 independent cell passages). * p < 0.05 from one-way ANOVA test.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Quantitative RT-PCR, Two Tailed Test, Expressing, shRNA, Generated

Journal: Oncogene

Article Title: A TGFβ-PRMT5-MEP50 Axis Regulates Cancer Cell Invasion through Histone H3 and H4 Arginine Methylation Coupled Transcriptional Activation and Repression

doi: 10.1038/onc.2016.205

Figure Lengend Snippet: A. Immunoblots for WDR5 and GAPDH from whole cell lysates of A549 treated with (+, blue bars) or without (−) TGFβ1 and with (+) or without (−) siRNA against WDR5. B. qRT-PCR of relative expression of Snail and Vimentin in A549 cells treated with (+, blue bars) or without (−) TGFβ1 and with (+) or without (−) OICR-9429, a small molecule inhibitor of the WDR5:H3 interaction. C. RT-qPCR as described in A except with A549 cells with (+, blue bars) or without (−) TGFβ1 and with (+) or without (−) expressing siRNA againstWDR5. D. H3R2me1 and H3K4me3 ChIP-qPCR experiments of up-regulated genes (Snail1 and Vim) in GFP kd and PRMT5 kd A549 cells treated with (+, blue bars) or without (−) TGFβ1. E. H3R2me1 and H3K4me3 ChIP-qPCR experiments of up-regulated genes (Snail1 and Vim) in A549 treated with (+, blue bars) or without (−) TGFβ1 and with (+) or without (−) OICR-9429. F. ChIP-qPCR as described in A except with A549 cells with (+, blue bars) or without (−) TGFβ1 and with (+) or without (−) expressing siRNA against WDR5. G. Model depicting the TGFβ1-PRMT5-MEP50 histone code axis involved in lung cancer metastasis progression. TGFβ1 stimulates PRMT5 protein expression and activity towards H4R3me2s (left, histone tail exaggerated for illustration), downregulating expression of cancer metastasis suppressor genes, and towards H3R2me1 (right), stimulating expression of EMT activator genes through recruitment of WDR5 and MLL to methylate H3K4me3. The targeted genes are central to regulation of cellular movement, EMT, and metastasis. Knockdown of PRMT5 or MEP50 can prevent this axis. A novel, specific, and highly potent PRMT5 inhibitor GSK591 also inhibits TGFβ1 induced cancer phenotypes with limited effect on survival of normal lung cells.

Article Snippet: Purified human PRMT5 (Sinobiological) and PRMT5 in complex with MEP50 were prepared as described and used as controls.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Activity Assay

Journal: Nature Communications

Article Title: Aberrant expression of CITED2 promotes prostate cancer metastasis by activating the nucleolin-AKT pathway

doi: 10.1038/s41467-018-06606-2

Figure Lengend Snippet: CITED2 forms a multimeric complex with nucleolin, p300, and PRMT5 subunits. a HEK293T cells were transfected with the Flag/SBP-CITED2 plasmid. CITED2-interacting proteins were precipitated using anti-Flag tag antibody or streptavidin, and identified by LC-MS/MS analyses. The proteins identified commonly in two precipitates are listed. b Prostate cancer cell lysates were immunoprecipitated with anti-CITED2 antibody or IgG, and the precipitates were immunoblotted with the indicated antibodies. c HEK293T cells were transfected with pcDNA or CITED2, and the cell lysates were subjected to FPLC. The FPLC elutes were immunoblotted with the indicated antibodies. The red box indicates a bigger-sized complex (600 to 700 kDa) that is formed by CITED2 overexpression. d In vitro binding analysis. Recombinant protein CITED2, PRMT5, NCL, and P300 were put together in a test tube. Proteins in tube were immunoprecipitated with indicated antibodies, and the precipitates were immunoblotted. Input levels were verified by electrophoresis and Coomassie staining. e Representative immunofluorescence images. PC3 cells were grown on coverslips, fixed with methanol, and stained with the indicated antibodies. All samples were stained with DAPI to visualize nuclei. The scale bar represents 20 μm. f The Flag/SBP-CITED2 constructs are shown in the top panel. HEK293T cells were cotransfected with one of the CITED2 constructs and Myc-PRMT5, HA-P300, and Flag/SBP-peptides were immunoprecipitated with anti-Flag and Myc-PRMT5 and HA-P300 were detected by western blotting. g HEK293T cells were transfected with the CITED2 constructs, and cell lysates were immunoprecipitated with anti-NCL and Flag/SBP-CITED2 peptides were detected by western blotting. h The Myc-PRMT5 constructs are shown in the top panel. HEK293T cells were cotransfected with one of the PRMT5 construct and Flag/SBP-CITED2, and Myc-peptides were immunoprecipitated with anti-Myc and Flag/SBP-CITED2 were detected by western blotting. All experiments were carried out at three distinct samples

Article Snippet: Human recombinant proteins of CITED2, PRMT5, P300, and NCL were purchased from Origene.

Techniques: Transfection, Plasmid Preparation, FLAG-tag, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Over Expression, In Vitro, Binding Assay, Recombinant, Electrophoresis, Staining, Immunofluorescence, Construct, Western Blot

Journal: Nature Communications

Article Title: Aberrant expression of CITED2 promotes prostate cancer metastasis by activating the nucleolin-AKT pathway

doi: 10.1038/s41467-018-06606-2

Figure Lengend Snippet: CITED2 is essential for post-translational modifications of NCL. a HEK293T cells were transfected with pcDNA or CITED2 and treated with Leptomycin B (200 nM), and the cell lysates were fractionated to cytosolic and nuclear components. The cell fractions were immunoblotted with the indicated antibodies. b HEK293T cells were cotransfected with Flag/SBP-NCL and CITED2 or si-CITED2. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with the indicated antibodies. c HEK293T cells were transfected with Myc-PRMT5 and/or si-PRMT5. Cell lysates were immunoprecipitated with anti-dimethyl arginine antibody and precipitated NCL was immunoblotted. d HEK293T cells were transfected with HA-p300. Cell lysates were immunoprecipitated with anti-acetyl lysine antibody and precipitated NCL was immunoblotted. e , f HEK293T cells were cotransfected with Flag/SBP-NCL and CITED2 or si-CITED2. Cell lysates were immunoprecipitated with anti-dimethyl arginine or anti-acetyl lysine antibody and precipitated NCL was immunoblotted. g , h HEK293T cells were cotransfected with the indicated plasmids and siRNAs. Cell lysates were immunoprecipitated with anti-dimethyl arginine or anti-acetyl lysine antibody and precipitated NCL was immunoblotted. i HEK293T cells were cotransfected with Flag/SBP-NCL and CITED2, and the cell lysates were fractionated to cytosolic and nuclear components. The cell fractions were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. j The ERG–CITED2–PRMT5/p300–NCL pathway in prostate cancer. ERG is upregulated due to the TMPRSS2–ERG gene fusion and transactivates the CITED2 gene in prostate cancer cells. Overexpressed CITED2 induces the methylation and acetylation of NCL in the nucleus by recruiting PRMT5 and p300, then modified NCL is translocated to the cytoplasm. All experiments were carried out at three distinct samples

Article Snippet: Human recombinant proteins of CITED2, PRMT5, P300, and NCL were purchased from Origene.

Techniques: Transfection, Immunoprecipitation, Methylation, Modification

Journal: Journal of Cellular and Molecular Medicine

Article Title: PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

doi: 10.1111/jcmm.14894

Figure Lengend Snippet: DNA and RNA nucleotide sequences

Article Snippet: To confirm the clinical relevance of PRMT5 expression, we first analysed the PRMT5 protein expression in clinical specimens from the human protein atlas ( http://www.proteinatlas.org ).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

doi: 10.1111/jcmm.14894

Figure Lengend Snippet: PRMT5 is profiled in pancreatic cancers and different pancreatic cancer cells. A, PRMT5 protein expression in pancreatic cancer tissues and normal pancreatic tissues was analysed through the human protein atlas ( http://www.proteinatlas.org ). Magnification, ×4; bars, 500 μm. Magnification, ×40; bars, 100 μm. B, Analysis of PRMT5 mRNA levels in 39 pairs of pancreatic cancer and non‐tumour tissues in Badea pancreas database. N = 39 for non‐tumour group, and N = 39 for tumour group. *** P < .001. C, Analysis of the TCGA database indicates that PRMT5 expression is correlated with patient’ overall survival. N = 32 for PRMT5‐low group, and N = 50 for PRMT5‐high group. P = .0345 was determined by log‐rank test. D, Analysis of the TCGA database indicates PRMT5 correlates with clinicopathological features. The results are presented by heat map (left panel) and box plot (right panel). N = 139 for pancreas adenocarcinoma ductal type group, N = 23 for pancreas adenocarcinoma other subtype group, and N = 4 for pancreas colloid (mucinous non‐cystic) carcinoma group. P = .0707 (E) Analysis of the TCGA database indicates PRMT5 is associated with stage in pancreatic cancer. The results are presented by heat map (left panel) and box plot (right panel). N = 20 for stage I group, N = 147 for stage II + III + IV group. * P < .05

Article Snippet: To confirm the clinical relevance of PRMT5 expression, we first analysed the PRMT5 protein expression in clinical specimens from the human protein atlas ( http://www.proteinatlas.org ).

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

doi: 10.1111/jcmm.14894

Figure Lengend Snippet: PRMT5 promotes cell proliferation in pancreatic cancer cells and tumorigenesis. A‐B, CCK‐8 assay showed that PRMT5 knockdown inhibited PaTu8988 and SW1990 cell growth rate (Student's t test:* P < .05). C‐D, Clone formation assays in PaTu8988 and SW1990 cells. PRMT5 knockdown inhibited cell clone formation (Student's t test:* P < .05). The number of clones with at least 50 cells per colony and strong, high dense staining was counted. The rates of colony formation were 48.33% and 17.67% in sh‐EGFP and sh‐PRMT5 PaTu8988 cells, and 35.67% and 10.67% in sh‐EGFP and sh‐PRMT5 SW1990 cells, respectively. E‐F, PaTu8988 cells with PRMT5 down‐regulation were injected (2.0 × 10 6 cells/site) subcutaneously into a mice, and the tumour volume was measured weekly (n = 5 mice). * P < .05. G‐H, CCK‐8 assay showed that ectopic PRMT5 re‐expression in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells promoted cell proliferation rate (Student's t test: * P < .05). I‐J, Clone formation assays in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells. Ectopic PRMT5 re‐expression in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells promoted cell clone formation (Student's t test: *** P < .001). The rates of number of colonies (defined as ≥50 cells) were 25.33% and 56.67% in PaTu8988 cells, 18.67% and 58.33% in SW1990 cells, respectively. K‐L, SW1990 cells with PRMT5 up‐regulation were injected (2.0 × 10 6 cells/site) subcutaneously into a mice, and the tumour volume was measured weekly (n = 5 mice), * P < .05

Article Snippet: To confirm the clinical relevance of PRMT5 expression, we first analysed the PRMT5 protein expression in clinical specimens from the human protein atlas ( http://www.proteinatlas.org ).

Techniques: CCK-8 Assay, Knockdown, Clone Assay, Staining, Injection, Expressing, Infection

Journal: Journal of Cellular and Molecular Medicine

Article Title: PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

doi: 10.1111/jcmm.14894

Figure Lengend Snippet: PRMT5 promotes cell migration and invasion in pancreatic cancer cells. A‐B, The migration changes were measured by transwell assay in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells (Student's t test: ** P < .01, *** P < .001). C‐D, After transfected with pHA‐Venus or pHA‐PRMT5 plasmid in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells, the migration changes were measured by transwell assay (Student's t test: ** P < .01). E‐F, The invasion changes were measured by transwell assay in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells (Student's t test: *** P < .001). The number of cells passing through the reconstituted basement membrane was 363.13 ± 26.25 and 152.54 ± 9.13 in sh‐EGFP and sh‐PRMT5 PaTu8988 cells, and 204.38 ± 18.38 and 89.54 ± 6.75 in sh‐EGFP and sh‐PRMT5 SW1990 cells, respectively. G‐H, After transfected with pHA‐Venus or pHA‐PRMT5 plasmid in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells, the invasion changes were measured by transwell assay (Student's t test: ** P < .01). Numbers of cells passing through the reconstituted basement membrane were 212.33 ± 12.54 and 403.13 ± 35.42 in PaTu8988 cells, 132.04 ± 8.29 and 212.21 ± 16.46 in SW1990 cells, respectively. I‐J, The protein levels of MMP2 and MMP9 were measured by Western blot

Article Snippet: To confirm the clinical relevance of PRMT5 expression, we first analysed the PRMT5 protein expression in clinical specimens from the human protein atlas ( http://www.proteinatlas.org ).

Techniques: Migration, Transwell Assay, Infection, Transfection, Plasmid Preparation, Membrane, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

doi: 10.1111/jcmm.14894

Figure Lengend Snippet: PRMT5 promotes EMT in pancreatic cancer cells. A, The protein levels of E‐cadherin, collagen I, β‐catenin and Vimentin were measured by Western blot in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells. B, After transfected with pHA‐Venus or pHA‐PRMT5 plasmid in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells, the protein levels of E‐cadherin, collagen I, β‐catenin and Vimentin were measured by Western blot. C‐D, Vimentin, β‐catenin and Collagen I mRNA levels were reduced while E‐cadherin mRNA level was increased in shPRMT5‐PaTu8988 and SW1990 cells. * P < .05, ** P < .01, *** P < .001. E‐F, Vimentin, β‐catenin and Collagen I mRNA levels were increased while E‐cadherin mRNA level was reduced in sh‐PRMT5 PaTu8988 and SW1990 cells transfected with pHA‐PRMT5 plasmid. * P < .05, ** P < .01, *** P < .001

Article Snippet: To confirm the clinical relevance of PRMT5 expression, we first analysed the PRMT5 protein expression in clinical specimens from the human protein atlas ( http://www.proteinatlas.org ).

Techniques: Western Blot, Infection, Transfection, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: PRMT5 promotes epithelial‐mesenchymal transition via EGFR‐β‐catenin axis in pancreatic cancer cells

doi: 10.1111/jcmm.14894

Figure Lengend Snippet: PRMT5 activates EGFR/AKT/β‐catenin signalling in pancreatic cancer cells. A, Wnt/β‐catenin and EGFR signalling relative proteins were detected by Western blot in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells. B, After transfected with pHA‐Venus or pHA‐PRMT5 plasmid in PaTu8988 and SW1990 sh‐PRMT5 stable infected cells, EGFR/AKT/β‐catenin signalling relative proteins were detected by Western blot. C, Pancreatic cancer cells were treated with EGFR inhibitor at 0, 10 μmol/L for 3 d. The expression of EGFR, p‐EGFR (Y1068) and PRMT5 was determined by Western blotting. D, The quantification of EGFR and p‐EGFR is shown. ** P < .01, *** P < .001. E, Wnt/β‐catenin and EGFR signalling relative proteins were detected by Western blot in PaTu8988 and SW1990 pHA‐PRMT5 stable infected cells treated with Erlotinib (10 μmol/L) for 3 d

Article Snippet: To confirm the clinical relevance of PRMT5 expression, we first analysed the PRMT5 protein expression in clinical specimens from the human protein atlas ( http://www.proteinatlas.org ).

Techniques: Western Blot, Infection, Transfection, Plasmid Preparation, Expressing