Figure 4.
Pevonedistat increases PD-L1 expression in ATLL cells. (A) Cell surface expression of PD-L1 in ATLL, ALK+ ALCL, and T-ALL cell lines treated with the indicated concentration of pevonedistat for 24 hours measured by flow cytometry. The y-axis represents MFI normalized by that of DMSO-treated cells. (B) mRNA expression of PD-L1 in KK1 and ST1 ATLL cells treated with pevonedistat for 24 hours as measured by the TaqMan gene expression assay. (C) Immunoblot analysis of PD-L1, pSTAT3, STAT3, CUL5, and α-tubulin in KK1 and ST1 ATLL cells treated with the indicated amount of pevonedistat for 24 hours. The arrowhead indicates the neddylated form of CUL5, which was decreased by using pevonedistat. (D) ST1 cells transduced with sgSTAT3 together with a GFP reporter or with a control sgAAVS1 were treated with pevonedistat for 24 hours. Cell surface expression of PD-L1 in the GFP-expressing cell populations was measured by flow cytometry. (E) Cell surface expression of PD-L1 in KK1, ST1, and Su9T01 ATLL cell lines treated with the indicated concentrations of pevonedistat and ruxolitinib for 24 hours was measured by flow cytometry. (F) Immunoblot analysis of PD-L1, pSTAT3, STAT3, CUL5, and α-tubulin in KK1, ST1, and Su9T01 ATLL cells treated with the indicated amounts of pevonedistat and ruxolitinib for 24 hours. The arrowhead indicates the neddylated form of CUL5. (G-H) Cell surface expression of PD-L1 in unmanipulated KK1, KK1 PD-L1 SV#7, and KK1 PD-L1 SV#15-12 cells treated with the indicated amount of pevonedistat (G) or with a combination of pevonedistat and ruxolitinib (H) for 24 hours was measured by flow cytometry. (I) Cell surface expression of PD-L1 in the mutated STAT3-transduced KK1 ATLL cells treated with the indicated amount of pevonedistat for 24 hours was measured by flow cytometry. (J) Immunoblot analysis of PD-L1, pSTAT3, STAT3, and GAPDH in the mutated STAT3-transduced KK1 ATLL cells. Error bars represent the mean with SEM of replicates. ∗P < .05; ∗∗P < .01; Welch 2-sample t test. All experiments were repeated at least twice. GFP, green fluorescent protein.

Pevonedistat increases PD-L1 expression in ATLL cells. (A) Cell surface expression of PD-L1 in ATLL, ALK+ ALCL, and T-ALL cell lines treated with the indicated concentration of pevonedistat for 24 hours measured by flow cytometry. The y-axis represents MFI normalized by that of DMSO-treated cells. (B) mRNA expression of PD-L1 in KK1 and ST1 ATLL cells treated with pevonedistat for 24 hours as measured by the TaqMan gene expression assay. (C) Immunoblot analysis of PD-L1, pSTAT3, STAT3, CUL5, and α-tubulin in KK1 and ST1 ATLL cells treated with the indicated amount of pevonedistat for 24 hours. The arrowhead indicates the neddylated form of CUL5, which was decreased by using pevonedistat. (D) ST1 cells transduced with sgSTAT3 together with a GFP reporter or with a control sgAAVS1 were treated with pevonedistat for 24 hours. Cell surface expression of PD-L1 in the GFP-expressing cell populations was measured by flow cytometry. (E) Cell surface expression of PD-L1 in KK1, ST1, and Su9T01 ATLL cell lines treated with the indicated concentrations of pevonedistat and ruxolitinib for 24 hours was measured by flow cytometry. (F) Immunoblot analysis of PD-L1, pSTAT3, STAT3, CUL5, and α-tubulin in KK1, ST1, and Su9T01 ATLL cells treated with the indicated amounts of pevonedistat and ruxolitinib for 24 hours. The arrowhead indicates the neddylated form of CUL5. (G-H) Cell surface expression of PD-L1 in unmanipulated KK1, KK1 PD-L1 SV#7, and KK1 PD-L1 SV#15-12 cells treated with the indicated amount of pevonedistat (G) or with a combination of pevonedistat and ruxolitinib (H) for 24 hours was measured by flow cytometry. (I) Cell surface expression of PD-L1 in the mutated STAT3-transduced KK1 ATLL cells treated with the indicated amount of pevonedistat for 24 hours was measured by flow cytometry. (J) Immunoblot analysis of PD-L1, pSTAT3, STAT3, and GAPDH in the mutated STAT3-transduced KK1 ATLL cells. Error bars represent the mean with SEM of replicates. ∗P < .05; ∗∗P < .01; Welch 2-sample t test. All experiments were repeated at least twice. GFP, green fluorescent protein.

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