human pca c4 2 cell lines  (ATCC)

Journal: International Journal of Biological Sciences

Article Title: Loss of UFL1 confers enzalutamide resistance of prostate tumors by regulating METTL16-mediated m6A modification of EEF1A1 mRNA

doi: 10.7150/ijbs.124214

Figure Lengend Snippet: UFL1 is downregulated in ENZR CRPC. (A) Analysis of UFL1 level in human PCa samples from the TCGA-PRAD dataset. (B) Representative IHC staining images for UFL1 in PCa tissues. (C) Statistical analysis of UFL1 protein levels by IHC. (D) WB examination demonstrating UFL1 protein levels in fresh PCa tumors and paired normal tissues. (E-H) Correlation of UFL1 mRNA level with Gleason score (E), clinical stage (F), lymph node metastasis (G), and distant metastasis (H). (I-J) UFL1 levels in PCa cell lines. LNCaP-Parental, LNCaP-ENZR, C4-2B-Parental, and C4-2B-ENZR cells were procured and examined by qRT-PCR (I) and WB (J). (K) Representative IHC images of UFL1 staining in tumor sections from xenograft mice. (L) UFL1 expression in the public GSE44927 dataset. (M-N) Representative IHC images demonstrating UFL1 level in CSPC and CRPC tissues. (O) Examination of UFL1 expression in human PCa samples from GEO datasets. (P-Q) In the published PCa cohort ( GSE116918 ) and TCGA-PRAD dataset, reduced UFL1 expression was correlated with worse overall survival. Scale bar, 50 µm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human PCa cell lines (LNCaP and C4-2B) along with HEK293T cells were procured from the American Type Culture Collection (ATCC) and cultured per the supplier's protocols.

Techniques: Immunohistochemistry, Quantitative RT-PCR, Staining, Expressing

Journal: International Journal of Biological Sciences

Article Title: Loss of UFL1 confers enzalutamide resistance of prostate tumors by regulating METTL16-mediated m6A modification of EEF1A1 mRNA

doi: 10.7150/ijbs.124214

Figure Lengend Snippet: METTL16 facilitates PCa cell growth, survival, and ENZR. (A) Violin plots illustrating CERES scores for all METTL family members across 11 PCa cell lines. (B) Analysis of METTL16 expression in human PCa samples from GEO datasets. (C) Representative IHC staining images for METTL16 and a statistical graph of METTL16 protein levels. (D) METTL16 expression in PCa cell lines. LNCaP-Parental, LNCaP-ENZR, C4-2B-Parental, and C4-2B-ENZR cells were collected, and total lysates were analyzed by WB (D). (E-J) Cell proliferation assessment utilized CCK-8 assay and colony formation assay in LNCaP-ENZR and C4-2B-ENZR cells following transfection with siNC or METTL16 siRNA. (K-L) Cell viability determination occurred in the designated cell lines during ENZ treatment. LNCaP-ENZR and C4-2B-ENZR cells underwent transfection as specified and received graded ENZ doses over 3 days. Cell viability quantification employed CCK-8 assays. (M-N) Recovery effects of wild-type METTL16 and catalytically inactive METTL16 mutants on METTL16 knockdown-mediated cell proliferation inhibition in LNCaP-ENZR and C4-2B-ENZR cells. (O-P) Cell viability determination in specified cell lines under ENZ treatment. Scale bar, 50 µm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human PCa cell lines (LNCaP and C4-2B) along with HEK293T cells were procured from the American Type Culture Collection (ATCC) and cultured per the supplier's protocols.

Techniques: Expressing, Immunohistochemistry, CCK-8 Assay, Colony Assay, Transfection, Knockdown, Inhibition

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