Fig. 3

NFAT1 increases PD-L1 expression via upregulation of TNF expression in RCC cells. A IHC Images of NFAT1 and PD-L1 staining using TMA tissue sections (n = 96 RCC). The scale bars were shown as indicated. B and C. Heatmap (B) and dot plot (C) to show the correlation of IHC scores for the expression of the PD-L1 and NFAT1 proteins in RCC patient specimens. (r = 0.5021 for spearman correlation coefficients, P < 0.001). D The GEPIA web tool was searched for the correlation between the expression of PD-L1 and NFAT1 in mRNA levels in RCC samples. P values as indicated in the Fig. E-G. Western blot (E), qRT-PCR (F) and FACS analysis (G) of PD-L1 expression in renal cancer cells infected with shControl or shNFAT1s. GAPDH served as an internal reference. For qRT-PCR analysis, data presented as the mean ± SD of three independent experiments. ***, P < 0.001. H and I. Western blot (H) and qRT-PCR (I) analysis of PD-L1 expression in renal cancer cells infected with EV or NFAT1 plasmids. GAPDH served as an internal reference. For qRT-PCR analysis, data presented as the mean ± SD of three independent experiments. ***, P < 0.001. The differences were compared between shControl group and shNFAT1 group. J After 72 h of selection with puromycin, 5 × 10⁶ Renca cells infected with shControl or shNFAT1 were subcutaneously injected into the right dorsal flank of C57BL/6 mice. Mice with subcutaneous Renca tumors (n = 5/group) were treated with anti-PD-1 (200 μg) or nonspecific IgG for three times at day 2, 4, and 7. The mean of each group was compared with the mean of every other group. K Immunofluorescence staining analysis of the percentage of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells infiltrated in Renca tumors. Data are presented as the mean ± SD of five independent experiments (***, P < 0.001). L ChIP-qPCR of TNF in 786-O and ACHN cells. All data are shown as the mean values ± SD from three replicates. ns not significant; ***p < 0.001, unpaired t test. The difference was compared between IgG group and NFAT1 group, or shControl group and shNFAT1 group. M 786-O cells were infected or transfected with indicated constructs. Then these cells were treated with or without TNF-α neutralized antibody (50 pg/ml) for 24 h. qRT-PCR analysis of PD-L1 expression in 786-O and ACHN cells. GAPDH served as an internal reference. Data presented as the mean ± SD of three independent experiments. ***, P < 0.001. The mean of each group was compared with the mean of every other group.