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. 2020 May 1;11:2156. doi: 10.1038/s41467-020-15547-8

Fig. 7. RAMS11 binds to chromobox 4 (CBX4) to regulate expression of Top2α mRNA and protein.

Fig. 7

RNA immunoprecipitation (RIP) shows binding of RAMS11 to CBX4 and not negative control IgG in a LoVo and b SW620 cells. c RNA pull down of 5-Bromo-UTP full-length RAMS11 probe showing binding of CBX4 by Western blot in LoVo and SW620 cells. d–g Decreased binding of CBX4 and active histone mark H3K4me3 at TOP2α promoter with silenced RAMS11 expression in chromatin immunoprecipitation (ChIP) assay. IgG n = 2, CBX4 n > 3, H3K4me3 n > 2. h RIP showing increased binding of RAMS11 to CBX4 in HT29 RAMS11 overexpressing cells. i, j ChIP of CBX4 and H3K4me3 shows increased binding to TOP2α promoter in HT29 RAMS11 overexpressing cells. IgG n = 3, CBX4 n = 3, H3K4me3 n = 2. k, l ChIP of CBX4 and H3K4me3 in CRISPR KO cells with RAMS11 overexpression (OE) rescue at TOP2α promoter. IgG n = 2, CBX4 n = 2, H3K4me3 n = 3. m Protein expression of TOP2α and CBX4 in LoVo (top) and SW620 cell lines (bottom). Band intensities were quantified from the digital image in ImageJ and are shown normalized to the wild-type lane for each target. Samples derived from the same experiment and blots were processed in parallel. Fold change normalized expression to actin is shown below gel. All data are presented as mean values ± s.d, analyzed by two-tailed paired t-test. Experiments repeated more than two times. *p < 0.05 **p > 0.005, #p < 0.0005. Source data are provided as a Source Data File.