human pca cell line c4-2  (Procell Inc)

Journal: The Journal of Clinical Investigation

Article Title: EZH2 crosstalk with RNA methylation promotes prostate cancer progression through modulation of m 6 A autoregulation pathway

doi: 10.1172/JCI195840

Figure Lengend Snippet: ( A ) The m 6 A ELISA to measure the global m 6 A levels in PrEC and BPH-1 benign prostate cells, along with a panel of PCa cell lines. ( B ) Western blot to detect the EZH2 expression level in all cell types tested in A . ( C and D ) The m 6 A ELISA to measure the global m 6 A levels in 2 PCa cell lines upon EZH2 knockdown ( C ), or in PrEC cells with EZH2 overexpression ( D ). ( E ) Representative fluorescence images to show the RNA m 6 A staining in control and EZH2-deficient C4-2 cells. Endogenous EZH2 were costained by anti-EZH2 antibody, and the nuclei were visualized by DAPI (Scale bar: 20 μm). ( F ) The m 6 A ELISA to measure the global m 6 A levels in C4-2 cells treated with a series of EZH2 inhibitors. For DZNeP, GSK126, and EPZ6438, a concentration of 5 μM was used. For all the MS drugs, a concentration of 1 μM was used. ( G ) Scatter plot showing the m 6 A methylation in C4-2 cells upon EZH2 depletion, with siEZH2 hypomethylated sites in blue and hypermethylated sites in red. ( H ) Violin plot to show the differential m 6 A ratio upon EZH2 knockdown in C4-2 cells. ( I ) The distribution of EZH2-affected m 6 A sites across the transcript. ( J ) Gene Ontology enrichment analysis of genes with hypomethylated m 6 A sites upon EZH2 knockdown in C4-2 cells. Statistical significance was assessed using the hypergeometric test with P values corrected for multiple testing with the g:SCS method using g:Profiler. ( K ) The m 6 A CUT&RUN-qPCR analysis to validate the EZH2-affected m 6 A sites in each indicated transcript of C4-2 cells. One-way ANOVA followed by Dunnett’s multiple-comparison test was used for statistical analysis in A , C , F , and K . Two-tailed Student’s t test was used in D .

Article Snippet: Human PCa cell line C4-2 was provided as a gift from Leland Chung (Cedars-Sinai, Los Angeles, California, USA), while PC-3, 22RV1, C4-2B, DU145, LNCaP, abl, and VCaP were purchased from ATCC.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Knockdown, Over Expression, Fluorescence, Staining, Control, Concentration Assay, Methylation, Comparison, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: EZH2 crosstalk with RNA methylation promotes prostate cancer progression through modulation of m 6 A autoregulation pathway

doi: 10.1172/JCI195840

Figure Lengend Snippet: ( A ) Western blot to detect the expression change of 11 common m 6 A mediators upon EZH2 suppression in 2 PCa cell lines. ( B ) Genome browser tracks to show the MeRIP-seq data at each indicated loci in C4-2 and PrEC cells, with the peaks around the stop codon and 3′-UTR regions being highlighted. ( C ) The m 6 A CUT&RUN-qPCR assay in 2 PCa cell lines to show the m 6 A enrichment in each indicated transcript. ( D ) Schematic of luciferase reporter constructs. Fragments of METTL14 or WTAP containing 3 predicted m 6 A consensus motifs were cloned downstream of the luciferase coding sequence (labeled as “WT”). In the mutant constructs (“Mut”), the adenosines at these m 6 A consensus sites were substituted with thymidines to disrupt potential m 6 A deposition and YTHDF1 binding. Fluc, firefly luciferase; Rluc, Renilla luciferase. ( E ) Luciferase reporter assay showing relative activity of WT versus mutant constructs in control, EZH2-deficient, or YTHDF1-deficient C4-2 cells. ( F ) Western blot to detect the change of METTL14 and WTAP proteins upon YTHDF1 suppression in 2 PCa cell lines. ( G and H ) Control, EZH2-, and YTHDF1-deficient C4-2 ( G ) and PC-3 ( H ) cells were treated with or without Proteasome inhibitor MG-132, followed by Western blot analysis to detect the change of METTL14 and WTAP proteins. Graph showing the relative METTL14 and WTAP protein levels in each indicated group based on 3 biologically independent experiments. ( I ) General overview of the site-specific RNA targeting using dCas13b-YTHDF1N fusion protein, which can trigger the assembly of translation machinery. Created with BioRender.com. ( J and K ) YTHDF1-deficient PCa cells were transfected with dCas13b-YTHDF1N and gRNAs targeting METTL14 ( J ) or WTAP ( K ), followed by Western blot analysis to measure the expression change of METTL14 and WTAP, respectively. Since the anti-YTHDF1 antibody we used cannot detect YTHDF1N, the anti-Flag antibody was utilized to capture the dCas13b-YTHDF1N proteins. One-way ANOVA followed by Dunnett’s multiple-comparison test was used for statistical analysis in E , G , and H . Two-tailed Student’s t test was used in C .

Article Snippet: Human PCa cell line C4-2 was provided as a gift from Leland Chung (Cedars-Sinai, Los Angeles, California, USA), while PC-3, 22RV1, C4-2B, DU145, LNCaP, abl, and VCaP were purchased from ATCC.

Techniques: Western Blot, Expressing, Luciferase, Construct, Clone Assay, Sequencing, Labeling, Mutagenesis, Binding Assay, Reporter Assay, Activity Assay, Control, Transfection, Comparison, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: EZH2 crosstalk with RNA methylation promotes prostate cancer progression through modulation of m 6 A autoregulation pathway

doi: 10.1172/JCI195840

Figure Lengend Snippet: ( A and B ) Puromycylation assay was conducted in C4-2 cells undergoing EZH2 enzymatic inhibitor treatment (5 μM) ( A ) or YTHDF1 deficiency ( B ). Before WB analysis, the whole protein extracts were visualized by UV and shown in the left panel as reference. ( C and D ) Scatter plots showing expression changes of mRNA levels and RPFs between control and EPZ6438-treated ( C ) or YTHDF1-deficient ( D ) C4-2 cells. Genes are colored according to their regulation mode. ( E ) Venn diagram showing the overlap between downregulated genes from translation mode after EPZ6438 treatment or YTHDF1 knockdown (KD). ( F ) Gene enrichment analysis of the overlapping genes in E using Reactome pathways. Statistical significance was assessed using the hypergeometric test with FDR-corrected P values. Only the top 8 enriched pathways are presented. ( G ) RIP-qPCR assay in C4-2 cells to test the binding of YTHDF1 proteins to each mRNA candidate as indicated. ( H ) The m 6 A CUT&RUN-qPCR assay in C4-2 cells upon GSK126 or EPZ6438 treatment to show the m 6 A alterations in each indicated transcript. ( I ) RIP-qPCR assay in C4-2 cells upon GSK126 or EPZ6438 treatment to monitor the change of YTHDF1 binding to each mRNA candidate as indicated. ( J ) Cytoplasmic polysome patterns of DMSO-, GSK126- and EPZ6438-treated C4-2 cells. ( K ) Quantification of the ratio of each polysomal-bound mRNA candidate to the total cytoplasmic mRNA of its own. One-way ANOVA followed by Dunnett’s multiple-comparison test was used for statistical analysis in H , I , and K . Two-tailed Student’s t test was used in G .

Article Snippet: Human PCa cell line C4-2 was provided as a gift from Leland Chung (Cedars-Sinai, Los Angeles, California, USA), while PC-3, 22RV1, C4-2B, DU145, LNCaP, abl, and VCaP were purchased from ATCC.

Techniques: Expressing, Control, Knockdown, Binding Assay, Comparison, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: EZH2 crosstalk with RNA methylation promotes prostate cancer progression through modulation of m 6 A autoregulation pathway

doi: 10.1172/JCI195840

Figure Lengend Snippet: ( A ) Cytoplasmic polysome pattern of C4-2 cells in each group as indicated. ( B ) Quantification of the ratio of indicated polysomal-bound mRNAs to the total cytoplasmic mRNA of their own. ( C ) Western blot to detect the expression of CCNB1, RAP1A, METTL14, and WTAP in each group of 2 PCa cell lines as indicated. ( D ) Cell viability assay to assess the proliferative capacity of GSK126- or EPZ6438-treated PCa cells (5 μM for each) overexpressing YTHDF1. ( E ) Colony formation assay was performed in each group as indicated. Graphs showing the percentage of the area in each well covered by crystal violet–stained cell colonies. ( F ) GFP-labeled C4-2 cells in each condition were injected into zebrafish embryos. Tumor cell invasion was examined upon 3 days and images were taken under 4× magnification. Embryos exhibiting positive circulation signals were classified as “invaded.” Graph showing the mean fluorescence intensity (%), with individual data points representing each measurement. For each group, 3 zebrafish larvae were randomly selected, and 2 distinct regions per larvae were analyzed to measure fluorescence intensity. One-way ANOVA followed by Dunnett’s multiple-comparisons test was used in panels B , E , and F when multiple groups were compared. For comparisons involving only 2 groups, an unpaired 2-tailed Student’s t test was applied ( B , E , and F ). In D , statistical significance was assessed using an unpaired 2-tailed Student’s t test at the final timepoint.

Article Snippet: Human PCa cell line C4-2 was provided as a gift from Leland Chung (Cedars-Sinai, Los Angeles, California, USA), while PC-3, 22RV1, C4-2B, DU145, LNCaP, abl, and VCaP were purchased from ATCC.

Techniques: Western Blot, Expressing, Viability Assay, Colony Assay, Staining, Labeling, Injection, Fluorescence