PD-L1/ IRF4 expression are dependent on ERK and AKT signaling. (A) DEL and Karpas299 lines were infected with GRB2, SOSO1, or Ctrl sgRNAs, selected and induced to expression. Lysates were analyzed by immunoblotting for the indicated proteins. (B) Indicated ALCL cell lines were treated with MEK inhibitor PD0325901 (10 μM), and PI3K/mTOR dual inhibitor BEZ235 (10 nM) for 24 hours, surface PD-L1 expression was measured by flow cytometry. Error bars denote SEM of triplicates. (C) DEL and Karpas299 cells were treated with MEK inhibitor PD0325901 and PI3K/mTOR dual inhibitor BEZ235 at indicated concentrations for 48 hours. Lysates were analyzed by immunoblotting for the indicated proteins. (D) DEL and Karpas299 cells were treated with MEK inhibitor PD0325901 and PI3K/mTOR dual inhibitor BEZ235 at indicated concentrations for 48 hours. IRF4 expression was measured by intracellular flow cytometry and normalized to DMSO controls. Error bars denote SEM of triplicates. (E) DEL and Karpas299 cells were treated with MEK inhibitor PD0325901 (1 μM) for 4 hours. IRF4 gene expression was measured by real-time PCR. Error bars denote SEM of triplicates. (F) DEL and Karpas299 cells transduced with WT IRF4 or with a control vector were treated with MEK inhibitor PD0325901 at indicated concentrations for 24 and 48 hours. Surface PD-L1 expression in treated and untreated (DMSO) cells were measured by flow cytometry. The relative PD-L1 MFI was normalized to untreated (DMSO) cells. Error bars denote SEM of triplicates. **P < .01. (G) Karpas299 cells were induced to express MTOR or Ctrl sgRNAs along with GFP. Intracellular IRF4 and surface PD-L1 expression in uninfected (GFP⁻) cells and sgRNA infected (GFP⁺) cells were measured by flow cytometry. The relative PD-L1 and IRF4 MFI was normalized to the uninfected (GFP⁻) cells. Error bars denote SEM of 4 repeats. P < .05 for all the data.