Figure 5.
Figure 5. Regulatory network for IRF4/BATF3 expression in ALK+ ALCL. (A) Indicated ALCL cell lines were treated with ALK inhibitor crizotinib for 24 hours at indicated concentrations and IRF4 expression was measured by intracellular flow cytometry and normalized to DMSO controls. (B) Indicated ALCL cell lines were treated with ALK inhibitor crizotinib for 24 hours at indicated concentrations. Lysates were analyzed by immunoblotting for the indicated proteins. (C) Karpas299 cells treated with ALK inhibitor crizotinib (300 nM) for 24 hours or untreated. Chromatin immunoprecipitation from indicated antibodies was subjected to real-time PCR analysis for PD-L1 locus. (D) ALCL lines were induced to express indicated sgRNAs along with GFP. Intracellular IRF4 expression in uninfected (GFP−) cells and sgRNA-infected (GFP+) cells were measured by flow cytometry. The relative IRF4 MFI was normalized to the uninfected (GFP−) cells. (E) 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. (F) DEL and Karpas299 cells transduced with WT IRF4 or with a control vector were induced to express control or indicated sgRNAs along with GFP. Surface PD-L1 expression in sgRNA uninfected (GFP−) cells and sgRNA-infected (GFP+) cells were measured by flow cytometry. The relative PD-L1 MFI of sgRNA-infected (GFP+) cells was normalized to sgRNA uninfected (GFP−) cells. Error bars denote SEM of triplicates. *P < .05; **P < .01; n.s indicates no statistically significant difference. (G) DEL and Karpas299 lines were infected with STAT3 or Ctrl sgRNAs, selected and induced to expression. Lysates were analyzed by immunoblotting for the indicated proteins. All error bars denote SEM of triplicates.

Regulatory network for IRF4/BATF3 expression in ALK+ ALCL. (A) Indicated ALCL cell lines were treated with ALK inhibitor crizotinib for 24 hours at indicated concentrations and IRF4 expression was measured by intracellular flow cytometry and normalized to DMSO controls. (B) Indicated ALCL cell lines were treated with ALK inhibitor crizotinib for 24 hours at indicated concentrations. Lysates were analyzed by immunoblotting for the indicated proteins. (C) Karpas299 cells treated with ALK inhibitor crizotinib (300 nM) for 24 hours or untreated. Chromatin immunoprecipitation from indicated antibodies was subjected to real-time PCR analysis for PD-L1 locus. (D) ALCL lines were induced to express indicated sgRNAs along with GFP. Intracellular IRF4 expression in uninfected (GFP−) cells and sgRNA-infected (GFP+) cells were measured by flow cytometry. The relative IRF4 MFI was normalized to the uninfected (GFP−) cells. (E) 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. (F) DEL and Karpas299 cells transduced with WT IRF4 or with a control vector were induced to express control or indicated sgRNAs along with GFP. Surface PD-L1 expression in sgRNA uninfected (GFP−) cells and sgRNA-infected (GFP+) cells were measured by flow cytometry. The relative PD-L1 MFI of sgRNA-infected (GFP+) cells was normalized to sgRNA uninfected (GFP−) cells. Error bars denote SEM of triplicates. *P < .05; **P < .01; n.s indicates no statistically significant difference. (G) DEL and Karpas299 lines were infected with STAT3 or Ctrl sgRNAs, selected and induced to expression. Lysates were analyzed by immunoblotting for the indicated proteins. All error bars denote SEM of triplicates.

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