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. 2023 Oct 25;9(43):eadg7752. doi: 10.1126/sciadv.adg7752

Fig. 5. Nimbolide triggers an innate immune response and up-regulates programmed death-ligand 1 (PD-L1) expression.

Fig. 5.

(A) Nimbolide treatment induces PARP1 trapping. UWB1 cells were treated with or without nimbolide (1 μM for 48 hours). The chromatin-bound fraction was isolated from these cells and was subject to immunoblotting experiments. (B) Nimbolide treatment induces DNA damage response. HeLa cells were treated with or without nimbolide (1 μM for 48 hours). The cell lysates were subject to immunoblotting experiments. (C) Nimbolide treatment induces the formation of cytosolic dsDNA and micronuclei. HeLa cells were treated with or without nimbolide (1 μM for 48 hours). Arrows indicate cytosolic dsDNA and micronuclei. (D) Nimbolide treatment induces the phosphorylation of TBK1. HeLa cells were treated with or without nimbolide (1 μM for 48 hours). The level of pS172 TBK1 (p-TBK1, green) was detected using the immunofluorescence assay. (E) Nimbolide treatment induces the phosphorylation of IRF3. HeLa cells were treated with or without nimbolide (1 μM for 48 hours). The level of pS396 IRF3 (p-IRF3, green) was detected using the immunofluorescence assay. (F) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses of IFN-β, CXCL10, or CCL5 in HeLa cells treated with or without nimbolide (1 μM for 48 hours). (G) Nimbolide treatment induces the expression of PD-L1. UWB1 cells were treated with or without nimbolide (1 μM for 48 hours). PD-L1 expression was detected using the immunoblot assay. (H) RNF114-KO abrogates the nimbolide-induced TBK1 phosphorylation. Control (RNF114-WT) and RNF114-KO HeLa cells were treated with or without nimbolide (1 μM for 48 hours). The whole-cell lysates were subject to immunoblot experiments. (I) PARP1-KO abrogates the nimbolide-induced TBK1 phosphorylation. Control (PARP1-WT) and PARP1-KO HeLa cells were treated with or without nimbolide (1 μM for 48 hours). The whole-cell lysates were subjected to immunoblot experiments. Scale bars, 10 μm. cGAS, cyclic guanosine 3′,5′-monophosphate–adenosine 5′-monophosphate synthase; DAPI, 4′ynthase; DAPI, nosine 3′,le; DMSO, dimethyl sulfoxide. pIRF3, phosphorylated IFN regulatory factor 3.