Fig. 5

JMJD6 enhances HOTAIR expression to maintain the stemness of LCSCs and promotes their radioresistance. A Quantitative analysis of JMJD6 and BRD4 expression in liver cancer and adjacent normal tissues measured by immunohistochemistry. *p < 0.05 compared with adjacent normal tissues. B The expression of JMJD6, BRD4 and ERK2 (MAPK1) in Hep3B and Huh7 CSCs with JMJD6 silencing or BRD4 silencing detected by Western blot, relative to GAPDH. *p < 0.05 compared with shCtl-treated cells. C The stemness of LCSCs with JMJD6 silencing or BRD4 silencing detected by microsphere formation assay. *p < 0.05 compared with shCtl-treated cells. D After exposure to X-ray, the cell colony formation in the presence of shJMJD6 or shBRD4, *p < 0.05 compared with shCtl-treated cells. E The enrichment of JMJD6 and BRD4 in the promoter region of HOTAIR after JMJD6 silencing, as examined by ChIP assay, relative to Input. The relative enrichment on the Y-axis represents JMJD6 or BRD4/IgG. *p < 0.05 compared with shCtl-treated cells. F Detection of expression of JMJD6 and LSD1 in cells following silencing of JMJD6, *p < 0.05 compared with shCtl-treated cells. G The expression of ERK2 (MAPK1) and JMJD6 after JMJD6 depletion or in combination with HOTAIR overexpression, as examined by Western blot and RT-qPCR. *p < 0.05 compared with shCtl + oe-NC-treated cells. #p < 0.05 compared with shJMJD6 + oe-NC-treated cells. H The microsphere formation assay was adopted to measure LCSC stemness after JMJD6 depletion or in combination with HOTAIR overexpression. *p < 0.05 compared with shCtl + oe-NC-treated cells. #p < 0.05 compared with shJMJD6 + oe-NC-treated cells. I Cell colony formation after JMJD6 depletion or in combination with HOTAIR overexpression following 6 Gy X-ray treatment examined by clonogenic assay. *p < 0.05 compared with shCtl + oe-NC-treated cells. #p < 0.05 compared with shJMJD6 + oe-NC-treated cells. J, RIP assay validated the interaction of JMJD6/BRD4 complex with LSD1 (relative enrichment in Y-axis represents IP-LSD1/IgG, * p < 0.05 compared with IgG group. Measurement data were expressed as mean ± standard deviation. Results between two groups were compared by unpaired t test. The data comparison between multiple groups was performed by one-way ANOVA with Tukey’s post-hoc test. Cellular experiments were repeated three times