Fig. 2: JunD-dependent RPS6KA2 transcription mediates BETi resistance.

a Western blotting was performed to detect JunD protein levels, MDA-MB-231 and BT549 cells were treated with DMSO or JQ1 (1 μM) for 0, 12 and 24 h. b Photograph depicted the potential JunD binding site in the enhancer region of RPS6KA2 gene. Wild-type and mutant RPS6KA2 gene enhancer luciferase plasmids are shown. c Luciferase assays were performed in MDA-MB-231 and BT549 cells transfected with shRNA for JunD and control, in the presence of DMSO or JQ1 (1 μM) for 6 h. Data are reported as mean ± SD. d Luciferase assays were performed in HEK293T cells transfected with wild-type or mutant RPS6KA2 enhancer luciferase construct with or without JUND co-transfection. HEK293T cells were treated with DMSO or JQ1 (1 μM) for 6 h. Data are reported as mean ± SD. e Chromatin immunoprecipitation (ChIP)-qPCR assay was executed in JUND-knockdown BLBC cells and their vector controls treated with DMSO or JQ1 (1 μM) for 6 h. ‘SP’ indicates specific primers of ChIP for RPS6KA2 gene enhancer and ‘NSP’ indicates non-specific primers that recognize the region downstream of the 3’ end of the gene. f Western blotting was performed to detect the expression levels of JunD and RSK3 in control and JUND-knockdown clones of MDA-MB-231 and BT549. g JUND or RPS6KA2-knockdown MDA-MB-231 and BT549 cells as well as their vector control cells were treated with DMSO or JQ1 (1 μM) for 48 h, and luminescent cell viability assays were done to detect the killing effects (**P < 0.01, one-way ANOVA). h Measured tumoursphere formation in JUND or RPS6KA2-knockdown MDA-MB-231 and BT549 cells as well as their vector controls. The cells were also treated with DMSO or JQ1 (1 μM). Statistical data of numbers of tumoursphere were shown (*P < 0.05, **P < 0.01; one-way ANOVA). i Rescued expression of RSK3 in control or JUND-knockdown BLBC cells which were treated with DMSO or JQ1 (1 μM) for 48 h. Cell viability was measured by CellTiter-Glo^® luminescent viability assay (**P < 0.01, ***P < 0.001; one-way ANOVA). j Western blotting was done to examine the expression levels of JunD and RSK3 in control or JUND-overexpressing clones of MDA-MB-231 and BT549. k JUND-overexpressing MDA-MB-231 and BT549 cells as well as their vector control cells were treated with JQ1 (1 μM) for 48 h, cell viability was detected by CellTiter-Glo^® luminescent viability assay. Statistical data (mean ± SD) are shown (**P < 0.01, ***P < 0.001; one-way ANOVA). Source data are provided as a Source Data file.