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: The following primary antibodies were used: AHR Rabbit mAb (clone D5S6H, CST #83200, RRID: AB_2800011), ARNT Rabbit mAb (clone D28F3, CST #5537, RRID: AB_10694232), HLA-DRA polyclonal Rabbit Ab (Boster #A01195), β-Tubulin Mouse mAb (clone C66, Abmart # M20005 , RRID: AB_2920648), and β-Actin Rabbit mAb (clone 13E5, CST #4970, RRID: AB_2223172).

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