Fig. 3: Fumarate facilitates PD-L1 expression via HIF-1α.

A Western blot analysis was performed to assess the protein levels in four human tumor cell lines (non-small cell lung cancer H1299, melanoma A375, liver cancer HCCLM3, breast cancer MDA-MB-231) and two mouse tumor cell lines (colorectal cancer CT26, breast cancer 4T1) following treatment with 50 μM DMF for 12 h. B The expression levels of HIF-1α, HIF-2α, and PD-L1 in RCC4 cells with or without transfection of VHL-expressing plasmids, were examined by Western blot after treatment with DMF (50 μM, 12 h). C Western blot and qRT-PCR analyses were performed to assess the PD-L1 levels in RCC10 and RCC4 cells treated with DMF, in the absence and presence of 5 μM YC-1, an inhibitor of HIF-1α. ***P < 0.001. D Western blot and qRT-PCR analyses were performed to assess PD-L1 levels in RCC10 and RCC4 cells treated with DMF, with or without 50 μM TC-S 7009, an inhibitor of HIF-2α. ns nonsignificant, **P < 0.01, ***P < 0.001. E Western blot analyses were performed to assess the PD-L1 levels in RCC4 cells treated with FHIN1, both in the absence and presence of 5 μM YC-1, which is an inhibitor of HIF-1α. F Western blot analyses were performed to assess PD-L1 levels in RCC4 cells treated with FHIN1 with or without 50 μM TC-S 7009, which is an inhibitor of HIF-2α. G After transfection with two siRNA fragments designed to knock down HIF-1α expression, RCC4 cells were treated with DMF, followed by the detection of PD-L1 expression levels through western blot. H After transfection of RCC4 cells with two HIF-2α-knockdown siRNAs, treatment with DMF was followed by detection of PD-L1 expression using western blot. I After transfection with two siRNA fragments designed to knockdown HIF-1α expression, RCC4 cells were treated with FHIN1, followed by the detection of PD-L1 expression levels through western blot. J After transfection of RCC4 cells with two HIF-2α-knockdown siRNAs, treatment with FHIN1 was followed by detection of PD-L1 expression using western blot. K Following pretreatment with YC-1 or TC-S 7009, the membrane PD-L1 expression in DMF-treated RCC4 cells was analyzed using flow cytometry. Representative histograms and summarized mean fluorescent intensity (MFI) are shown. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by Student’s t-test. ns nonsignificant, **P < 0.01.