Fig. 4.

Knockdown of KMT2C/2D reduces WT and Y537S ERα transcriptional activity and breast cancer cell growth. a HeLa cells grown in phenol red-free, charcoal-stripped media were co-transfected with pERE-E1b-luc, YFP-tagged Y537S ERα, and different siRNAs (25 nM each for 50 nM total). Cell lysates were assayed for luciferase activity (RLU). Data are represented as mean ± SEM (n = 3); ***p < 0.001. NC#1 siRNA served as the negative control for KMT2D targeting siRNA, while Control-A siRNA pool served as the negative control for KMT2C targeting siRNA pool. b Transfection of siRNAs targeting KMT2C and KMT2D reduced expression of the pERE-E1b-luc reporter, as compared to non-targeting siRNAs (siControl), in HeLa cells co-transfected with YFP-tagged WT or Y537S ERα vectors, but not an YFP-tagged ESR1-YAP1 fusion. Luciferase activity was measured as in Fig. 1b. Data are represented as mean ± SEM (n = 3); **p < 0.01; ***p < 0.001. c Knockdown of KMT2C and KMT2D in lentiviral transduced stably expressing WT or Y537S MCF-7 cells results in reduced anchorage-independent growth in soft agar and confers sensitivity to anti-estrogens. siRNAs (same as above) were transfected into the two cell lines at a final concentration of 100 nM (50 nM each), and then re-plated in 24 well plates 24 h later. After 1 week with either vehicle or anti-estrogen (100 or 1000 nM 4-hydroxytamoxifen (Tam) or ICI) treatment, colonies formed in soft agar were counted and quantified. Data are represented as mean ± SEM (n = 4); *p < 0.05; **p < 0.01; ***p < 0.001