Fig. 5: STAT6 governs chemotherapy-induced GAS6 transcription in Ewing sarcoma.

a A cartoon illustration to reveal the involvement of transcription factors in mediating chemotherapy-induced GAS6 transcription in Ewing sarcoma. b, c IB analysis of WCL from control or STAT6-depleted MHH-ES-1 cells treated with or without indicated doses of SN-38 (b) or etoposide (c) for 24 h. d RT-PCR analyses of mRNA expression changes of GAS6 in control or STAT6-depleted MHH-ES-1 cells treated with SN-38 (1 μM) for 24 h. p values are labeled and represent differences between experimental groups compared to the control group (one-way ANOVA test). e IB analysis of both WCL and BCA-precipitated culture media from control or STAT6-depleted MHH-ES-1 cells treated with SN-38 (1 μM) for 24 h. Error bars were calculated as mean ± SD, n = 3 (experimental triplicates). *p < 0.05 represents differences between the experimental groups compared to the control group (one-way ANOVA test). f upper: an illustration of predicted STAT6 binding sites on human GAS6 promoter. Lower: representative PCR analysis for STAT6 cut&run from MHH-ES-1 cells treated with control or 1 μM SN-38 for 24 h. g Cell viability assays using control or STAT6-depleted MHH-ES-1 cells treated with the indicated dose of SN-38 (1 μM) for 48 h. Error bars were calculated as mean ± SD, n = 3 (experimental triplicates). *p < 0.05 represents differences between the experimental groups compared to the control group (one-way ANOVA test). *p value of each point of g: sgSTAT6/sgctrl: 0.0003&0.0008 (30 μM), 0.0001&0.0007 (100 μM), 0.0001&0.0015 (300 μM). h Cell viability assays using control or STAT6-depleted MHH-ES-1 cells treated with the indicated dose of SN-38 (1 μM) for 48 h. Where indicated, STAT6-depleted MHH-ES-1 cells were pre-treated with GAS6-conditioned media for 24 h prior to SN-38 treatment. Error bars were calculated as mean ± SD, n = 3 (experimental triplicates). *p < 0.05 represents differences between the experimental groups compared to the control group (one-way ANOVA test). *p value of each point of h: sgSTAT6-GAS6-conditioned media/sgctrl-normal media: <0.0001 (300 μM), sgSTAT6-GAS6-conditioned media/ sgSTAT6-normal media: 0.0118 (300 μM). WB data presented in this figure are representative data from biological duplicates.