Fig. 3

STAT1 is required for A^IFN−γ-induced PD-L1 expression in cancer cells and elF4F inhibition blocks A^IFN−γ-mediated PD-L1 expression in cancer cells. A Analysis of pSTAT1, STAT1 and β-actin protein levels in MC38^wt cells treated with acidic media and/or IFN-γ (10 ng ml⁻¹) for 12 h and 24 h as determined using Western blotting (B) Densitometry of pSTAT1/STAT1 Western blotting of A. C-F MC38^wt cells were transfected with a control (siCTL) or Stat1-specific siRNA and treated for 24 h with acidic media ± IFN-γ (10 ng ml⁻¹) before the assessment of relative (C) Stat1 and (D) Pdl1 mRNA expression normalized to Gapdh, Aldolase and β-actin (n = 3) and (E) cell surface PD-L1 expression (n = 3) using qRT-PCR and flow cytometry, respectively. (A, H) The two signals per condition represent two independent samples (n = 2); two independent experiments showed similar results. Statistics: Tukey’s multiple comparison test. F Western blot analyses of pSTAT1, STAT1, PD-L1 and β-actin levels were performed to determine the STAT1 knockdown efficiency (n = 2). Data are presented as the means ± SEM. N = neutral media, A = acidic media, N^IFN−γ = neutral media plus IFN-γ, A^IFN−γ = acidic media plus IFN-γ. G Schematic representation of the eukaryotic translation initiation complex elF4F composed of elF4G, elF4E and elF4A (inhibited by silvestrol) bound to the 5’UTR of the Stat1 mRNA. H Western blot analysis of elF4A1, elF4E and β-actin levels in MC38^wt cells treated with acidic media and/or IFN-γ (10 ng ml⁻¹) for 72 h (n = 2). Relative (I) Stat1 and (J) Pdl1 mRNA expression normalized to Gapdh, Aldolase and β-actin (n = 3) and (K) cell surface PD-L1 expression (n = 3) were determined in MC38^wt cells treated with acidic media and/or IFN-γ (10 ng ml⁻¹) in the presence of DMSO (control) or silvestrol (30 nM) for 24 h. Data are presented as the means ± SEM. Statistics: Tukey’s multiple comparison test. N = neutral media, A = acidic media, N^IFN−γ = neutral media plus IFN-γ, A^IFN−γ = acidic media plus IFN-γ