Figure 3. DOT1L increases Snail, ZEB1 and ZEB2 expression to repress E-cadherin expression and breast CSC property.

(a) CDH1 mRNA levels in the indicated cell lines were analysed using qRT–PCR. For transient knockdown of DOT1L, five different siRNAs against DOT1L (siDOT1L, #1–5) or siCON were transfected into MDA-MB-231 cells (middle). MCF10A cells expressing DOT1L wild-type (DOT1L) or DOT1L siRNA (#2)-resistant mutant (DOT1L-si MUT) were transfected with DOT1L siRNA #2 or control siRNA for 48 h, and subjected to qRT–PCR for analysis of CDH1 expression (right). *P<0.05 versus controls (CON, siCON, CON/siCON) by Student's t-test. (b) Effect of DOT1L on CDH1 promoter activity. Cells were transfected with luciferase constructs of wild-type (CDH1 pro-WT) or E-box-mutant (CDH1 pro-MUT) CDH1 promoters for 24 h and the luciferase activity was measured. RLU, relative light units. *P<0.05 versus CON (Student's t-test). (c–e) Effects of DOT1L on the expression of CDH1 transcriptional regulators were examined using immunoblotting (c), qRT–PCR (d) and immunofluorescence staining (e). *P<0.05 versus CON or siCON (Student's t-test). Scale bars in e, 100 μm. (f) Binding by EMT-TFs to the CDH1 promoter region was analysed using ChIP–qPCR. *P<0.05 versus CON (Student's t-test). (g,h) Effect of EMT-TFs on DOT1L-induced cancer stemness. Cells were transfected with siRNAs against Snail, ZEB1 and ZEB2, and the CSC population was measured using flow cytometry. The knockdown of EMT-TFs was confirmed by immunoblotting (g). The FACS results were quantified and shown as a bar graph (h). * and ^†P<0.05 versus CON/siCON and DOT1L/siCON, respectively (Student's t-test). Error bars in a,b,d,f,h indicate the means±s.d. of experiments in triplicate.