Fig. 7: Chemotherapy promotes JAK1-pSQ to promote JAK1 activation.
From: Co-targeting JAK1/STAT6/GAS6/TAM signaling improves chemotherapy efficacy in Ewing sarcoma
a A cartoon illustration of the human JAK1 domain structure with the only S571Q motif labeled. b, c IB analysis of in vitro JAK1 kinase assay reactions by incubating indicated Flag-immunoprecipitated JAK1-FL (full-length) from control or SN-38 (1 μM for 24 h) treated MHH-ES-1 cells with bacterially purified GST-STAT6 (aa 633–801) proteins. Refer to the Method section for details. d–f IB analysis of Flag-IPs and WCL from HEK293T cells transfected with indicated DNA constructs and treated with either SN-38 (1 μM) or etoposide (10 μM) for 24 h. g, h IB analysis of in vitro JAK1 kinase assay reactions by incubating indicated Flag-immunoprecipitated JAK1-KM (kinase motif) from control or SN-38 (1 μM for 24 h) treated MHH-ES-1 cells with bacterially purified GST-STAT6 (aa 633–801) proteins. Refer to the Method section for details. i IB analysis of indicated MHH-ES-1 cells. j IB analysis of WCL from MHH-ES-1 cells depleted of endogenous JAK1 stably expressing indicated Flag-JAK1 treated with 1 μM SN−38 for 24 h. k Cell viability assays using indicated MHH-ES-1 cells treated with indicated doses of SN-38 for 48 h. Error bars were calculated as mean ± SD, n = 3 (experimental triplicates). *p < 0.05 (one-way ANOVA test). *p value of each point of k: WT/EV: <0.0001 (30 nM), WT/S571A: <0.0001 (30 nM). l IB analysis of in vitro JAK1 kinase assay reactions by incubating indicated Flag-immunoprecipitated JAK1-KM from control or SN-38 (1 μM for 24 h) treated MHH-ES-1 cells with bacterially purified GST-STAT6 (aa 633–801) proteins. Refer to the Method section for details. m IB analysis of Flag-IPs and WCL from HEK293T cells transfected with indicated DNA constructs and treated with either SN-38 (1 μM) for 24 h. n A cartoon model revealing chemotherapy induces DNA damage, leading to activation of DNA-damaging kinases including ATM, ATR, and DNAPK that subsequently phosphorylates JAK1 at S571Q motif to activate JAK1 intracellularly via releasing JAK1-JH2 mediated suppression of its JH1 kinase domain. WB data presented in this figure are representative data from biological triplicates.
