human melanoma cancer cell line a375  (ATCC)

Journal: Journal for Immunotherapy of Cancer

Article Title: Polycomb repressor complex 2 suppresses interferon-responsive MHC-II expression in melanoma cells and is associated with anti-PD-1 resistance

doi: 10.1136/jitc-2023-007736

Figure Lengend Snippet: High expression of PRC2 genes correlates with low expression of tumor-specific MHC class II genes. (A) Cluster analysis was performed using z-scores calculated from RNA sequencing expression data of patient with melanoma tumors gathered from The Cancer Genome Atlas. (B) mRNA expression of PRC2 genes ( EZH2 and JARID2 ) and T-cell genes ( CD3E, CD4, CD8A ) across T cell hi /(red boxes), Intermediate (blue boxes), and PRC2 hi (green boxes) patient clusters. Box plots show the median, first and third quartiles. The whiskers extend to the maxima and minima but no further than 1.5 times the IQR. Data were analyzed by analysis of variance followed by a Tukey’s post hoc test. ***p<0.001; ****p<0.0001. (C) Flow cytometry histograms representing live fractions of interferon-γ-stimulated (red histograms) human melanoma cell lines compared with unstimulated controls (gray histograms). Following staining with a live-dead dye, cells were labeled with fluorophore-conjugated HLA-DR antibodies. (D) qPCR analysis of human melanoma cell lines characterized for mRNA expression of the indicated genes of interest relative to GAPDH mRNA expression values for each cell line. Values were calculated as fold expression relative to A375 cells.CIITA, class II major histocompatibility complex transactivator II; EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; HLA-DR, human leukocyte antigen-DR ; JARID2, Jumoni and AT-rich interaction domain-containing protein; MHC, major histocompatibility complexes; mRNA, messenger RNA; PRC2, polycomb repressive complex 2; qPCR, quantitative polymerase chain reaction.

Article Snippet: Human melanoma cancer cell lines A375 (DMEM+10% FBS), SKMEL-5 (DMEM+10% FBS), SKMEL-28 (DMEM+10% FBS), COLO829 (RPMI+10% FBS), MeWo (MEM+10% FBS), SKMEL-1 (MEM+10% FBS), and CHL-1 (DMEM+10% FBS) were obtained from American Type Culture Collection.

Techniques: Expressing, RNA Sequencing, Flow Cytometry, Staining, Labeling, Immunopeptidomics, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Polycomb repressor complex 2 suppresses interferon-responsive MHC-II expression in melanoma cells and is associated with anti-PD-1 resistance

doi: 10.1136/jitc-2023-007736

Figure Lengend Snippet: Pharmacological inhibition of EZH2 upregulates interferon-induced major histocompatibility complexes expression. (A–B) Flow cytometry analysis of A375 and SKMEL-5 cell lines treated with the indicated EZH2 inhibitor for 5 days. Single cell suspensions were stained with fluorophore-conjugated antibodies HLA-DR. *p<0.05 and **p<0.01, one-sample t-test against a fold change of 1. (C) After stimulation with IFN-γ for 24–48 hours, whole cell protein lysates were extracted from A375, SKMEL-5, and COLO829 cells treated with DMSO or EZH2 inhibitor before being probed for the indicated proteins. Data shown represents three independent experiments. (D–E) Relative expression of EZH2, CIITA, and CD74 mRNA extracted from melanoma cell lines treated with GSK343 or tazemetostat and stimulated with IFN-γ for 24–48 hours. Samples were normalized to GAPDH mRNA and calculated as fold expression relative to the DMSO control. CIITA, class II major histocompatibility complex transactivator I; DMSO, dimethyl sulfoxide; EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; HLA-DR, human leukocyte antigen-DR; IFN, interferon; MFI, mean flourescence intensity; mRNA, messenger RNA; PD-L1, programmed death ligand 1.

Article Snippet: Human melanoma cancer cell lines A375 (DMEM+10% FBS), SKMEL-5 (DMEM+10% FBS), SKMEL-28 (DMEM+10% FBS), COLO829 (RPMI+10% FBS), MeWo (MEM+10% FBS), SKMEL-1 (MEM+10% FBS), and CHL-1 (DMEM+10% FBS) were obtained from American Type Culture Collection.

Techniques: Inhibition, Expressing, Flow Cytometry, Staining, Control, Immunopeptidomics

Journal: Journal for Immunotherapy of Cancer

Article Title: Polycomb repressor complex 2 suppresses interferon-responsive MHC-II expression in melanoma cells and is associated with anti-PD-1 resistance

doi: 10.1136/jitc-2023-007736

Figure Lengend Snippet: Tazemetostat treatment exposes accessible chromatin at regions containing putative IRF1 and STAT1 binding motifs in IFN-γ-stimulated CHL-1 cells. (A) CHL-1 cells were pretreated for 4 days with tazemetostat before 24 hours stimulation with IFN-γ. Cells were harvested and processed for assays for transposase-accessible chromatin with sequencing. Black bars represent predicted target sites for H3K27me3 marks, CpG islands, and promoters driving transcription of the indicated genes, based on the publicly available data from multiple tissue types and conditions in the UCSC database. Putative transcription factor (TF) binding sites for IFN-γ signaling (STAT1 and IRF4), based on predictions made using the https://molotool.autosome.org/ and a permissive p value setting of 0.0005. (B) mRNA expression assessed by qPCR using primers specific for the pIII and pIV 5’UTR. (C) Correlation matrix for qPCR data across seven cell lines (A375, SKMEL5, COLO829, SKMEL28, MEWO, SKMEL1, and CHL1) under interferon-stimulated conditions. CIITA, class II major histocompatibility complex transactivator II; DMSO, dimthyl sulfoxide; EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; IFN, interferon; JARID2, Jumoni and AT-rich interaction domain-containing; mRNA, messenger RNA; pIV, promoter IV; qPCR, quantitative polymerase chain reaction; USCS, University of California, Santa Cruz.

Article Snippet: Human melanoma cancer cell lines A375 (DMEM+10% FBS), SKMEL-5 (DMEM+10% FBS), SKMEL-28 (DMEM+10% FBS), COLO829 (RPMI+10% FBS), MeWo (MEM+10% FBS), SKMEL-1 (MEM+10% FBS), and CHL-1 (DMEM+10% FBS) were obtained from American Type Culture Collection.

Techniques: Binding Assay, Sequencing, Expressing, Immunopeptidomics, Real-time Polymerase Chain Reaction