wm115  (ATCC)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The aryl hydrocarbon receptor (AHR) drives human leukocyte antigen (HLA)-II expression in human melanoma

doi: 10.1186/s13046-026-03673-y

Figure Lengend Snippet: AHR and ARNT regulate basal HLA-II expression on cancer cells. A Western blot analysis confirming AHR or ARNT overexpression (OE) in A375, WM115, and SKMEL2 melanoma cell lines. Protein levels of AHR, ARNT, and HLA-DRA were assessed using anti-AHR (clone D5S6H), anti-ARNT (clone D28F3), and polyclonal anti–HLA-DRA antibodies, respectively, with β-tubulin detected using an anti–β-tubulin antibody (clone C66) as a loading control. B- C Flow cytometry analysis of surface pan–HLA-II expression in A375, WM115, and SKMEL2 cells overexpressing AHR or ARNT. Representative histograms from three independent experiments show pan–HLA-II expression, with dashed lines indicating the median of the empty vector control group ( B ). The gMFI was quantified across three independent experiments, normalized to the empty vector control group, and presented as fold change ( C ). D Western blot analysis of AHR, ARNT, and HLA-DRA protein levels in AHR or ARNT reconstituted A375 cells generated by reintroducing AHR or ARNT into respective KO cells. Protein levels of AHR, ARNT, and HLA-DRA were assessed using anti-AHR (clone D5S6H), anti-ARNT (clone D28F3), and polyclonal anti–HLA-DRA antibodies, respectively, with β-tubulin detected using an anti–β-tubulin antibody (clone C66) as a loading control. E - F Flow cytometry analysis of surface pan–HLA-II expression in AHR- or ARNT-reconstituted A375 cells. Representative histograms from three independent experiments show pan–HLA-II expression ( E ). The gMFI was quantified across three independent experiments, normalized to the NTC group, and presented as fold change ( F ). Surface pan–HLA-II expression was detected using APC anti-human HLA-DR, DP, DQ Antibody (clone Tü39) ( B - C and E - F ). Data are represented as mean ± SD ( C and F ). Statistical analysis by one-way ANOVA ( C ) and unpaired Student’s t-test ( F ); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. vec, empty vector control

Article Snippet: After 48 h of transfection, cells that had been transduced were selected using 0.7 μg/ml (A375) or 0.6 μg/ml (WM115) of puromycin (InvivoGen #ant-pr-1) for 3 days.

Techniques: Expressing, Western Blot, Over Expression, Control, Flow Cytometry, Plasmid Preparation, Generated

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The aryl hydrocarbon receptor (AHR) drives human leukocyte antigen (HLA)-II expression in human melanoma

doi: 10.1186/s13046-026-03673-y

Figure Lengend Snippet: Ligand-mediated activation of AHR regulates HLA-II expression on cancer cells. A Flow cytometry analysis of surface pan–HLA-II expression in A375, WM115, and SKMEL2 melanoma cells following 72-hour treatment with 2.5 μM GNF351 (AHR antagonist), 1 μM FICZ (AHR agonist), 100 ng/mL IFN-γ, or 0.1% DMSO (vehicle control). B - C Time-course analysis of pan–HLA-II surface expression in A375, WM115, and SKMEL2 cells following treatment with GNF351 (2.5 μM) or FICZ (1 μM) for 24, 48, or 72 hours. D - E pan–HLA-II expression in A375 cells at 72 hours post drug withdrawal following prior exposure to 2.5 μM GNF351 or 1 μM FICZ for 24, 48, or 72 hours. F - G Surface pan–HLA-II expression in WM115 ( F ) and SKMEL2 ( G ) cells at multiple timepoints post drug withdrawal, following treatment as in ( D - E ). H - I Surface pan–HLA-II expression in AHR or ARNT KO WM115 ( H ) and SKMEL2 ( I ) after 72-hour treatment with 1 μM FICZ or DMSO control. Surface pan–HLA-II expression was detected using APC anti-human HLA-DR, DP, DQ Antibody (clone Tü39). Representative histograms from three independent experiments show pan–HLA-II expression, with dashed lines indicating the median of the DMSO control group ( A , B and D ). The gMFI was quantified across three independent experiments, normalized to the DMSO control group ( C and E - G ) or to the mean of the two NTC sgRNAs with DMSO treatment ( H - I ), and presented as fold change. Data are represented as mean ± SD ( C and E - I ). Statistical analysis by one-way ANOVA (C and E); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: After 48 h of transfection, cells that had been transduced were selected using 0.7 μg/ml (A375) or 0.6 μg/ml (WM115) of puromycin (InvivoGen #ant-pr-1) for 3 days.

Techniques: Activation Assay, Expressing, Flow Cytometry, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The aryl hydrocarbon receptor (AHR) drives human leukocyte antigen (HLA)-II expression in human melanoma

doi: 10.1186/s13046-026-03673-y

Figure Lengend Snippet: AHR signaling modulates HLA-II expression via transcriptional regulation of CIITA. A - B Scatterplots showing differentially expressed genes in A375 cells with CRSIPR-mediated knockouts of AHR ( A ) or ARNT ( B ) compared to NTC cells. Significantly downregulated (blue) and upregulated (red) genes are defined by adjusted p < 0.0001 and |log2 fold change| > 0.5. The top 20 differentially expressed genes are indicated. C Correlation analysis of gene expression changes between AHR KO and ARNT KO groups. Each point represents a gene; commonly upregulated (red) and downregulated (blue) genes are highlighted, with CYP1A1 and HLA-II-related genes indicated. D - E Venn diagrams showing the overlap of significantly downregulated ( D ) and upregulated genes ( E ) between AHR KO and ARNT KO groups. F Heatmap showing the common significantly downregulated and upregulated genes in AHR KO, ARNT KO, and NTC samples of A375, with HLA-II genes and cellular stress and signaling response genes indicated. G - H KEGG pathway enrichment analysis of common significantly downregulated genes ( G ) and upregulated genes ( H ) in AHR KO and ARNT KO groups. I Scatterplot showing differentially expressed genes in WM115 cells treated with 1 μM FICZ for 72 h compared to the 0.1% DMSO control group. Significantly downregulated (blue) and upregulated (red) genes are defined by adjusted p < 0.01 and |log2 fold change| > 0.5. HLA-II-related genes are indicated. J Heatmap of differentially expressed genes in WM115 cells with FICZ versus DMSO control, with HLA-II-related genes annotated. K KEGG analysis of significantly upregulated genes in the FICZ-treated group compared to the DMSO control group of WM115. Statistical analysis by t-test (C) and hypergeometric test (G-H and K). PCC, Pearson correlation coefficient

Article Snippet: After 48 h of transfection, cells that had been transduced were selected using 0.7 μg/ml (A375) or 0.6 μg/ml (WM115) of puromycin (InvivoGen #ant-pr-1) for 3 days.

Techniques: Expressing, Gene Expression, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The aryl hydrocarbon receptor (AHR) drives human leukocyte antigen (HLA)-II expression in human melanoma

doi: 10.1186/s13046-026-03673-y

Figure Lengend Snippet: AHR-ARNT complex binds CIITA promoter II to drive HLA-II transcription. A - B Genome browser tracks showing gene CIITA mRNA expression (RNA-Seq) across the CIITA locus in A375 with sgNTC, sgAHR, or sgARNT (A) and in WM115 cells treated with 1 μM FICZ or 0.1% DMSO for 72 h (B). C - D ATAC-seq tracks showing chromatin accessibility at the CIITA locus in A375 cells with sgNTC, sgAHR, or sgARNT ( C ) and in WM115 cells treated with 1 μM FICZ or 0.1% DMSO for 72 h ( D ). E - F ChIP-seq tracks showing AHR or ARNT binding at the CIITA locus in SKMEL2 cells expressing empty vector control, 3×HA-AHR overexpression, or 3×HA-ARNT overexpression constructs, immunoprecipitated with anti-HA antibody (clone 1F5C6) ( E ) and in WM115 cells treated with 1 μM FICZ for 72 h, immunoprecipitated with IgG control (clone DA1E), anti-AHR (clone D5S6H), or anti-ARNT (clone D28F3) antibodies ( F ). G Enrichment analysis of transcription factor motifs in AHR- and ARNT-binding regions identified by ChIP-seq of SKMEL2 cells overexpressing 3×HA-tagged AHR or ARNT. Top-enriched motifs include canonical AHR response elements. H ChIP-qPCR quantification of AHR or ARNT occupancy at the CIITA promoter II (pII) region in SKMEL2 cells expressing empty vector control, 3×HA-AHR overexpression, or 3×HA-ARNT overexpression constructs. Enrichment is shown relative to input and normalized to the empty vector control. I Luciferase reporter assay using a construct containing the CIITA pII co-transfected with Renilla luciferase control into 293T cells expressing empty vector control, AHR overexpression, or ARNT overexpression constructs. vec, empty vector control. Data are represented as mean ± SD ( H - I ). Each dot represents one technical replicate from triplicate wells ( H - I ). Statistical analysis by one-way ANOVA ( H - I ); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. vec, empty vector control

Article Snippet: After 48 h of transfection, cells that had been transduced were selected using 0.7 μg/ml (A375) or 0.6 μg/ml (WM115) of puromycin (InvivoGen #ant-pr-1) for 3 days.

Techniques: Expressing, RNA Sequencing, ChIP-sequencing, Binding Assay, Plasmid Preparation, Control, Over Expression, Construct, Immunoprecipitation, ChIP-qPCR, Luciferase, Reporter Assay, Transfection