Fig. 4

Slug cooperates with PRMT5 and LSD1 to regulate the transcription of E-cadherin and vimentin. (A) ChIP and Re-ChIP experiments were done in MDA-MB-231 cells with the indicated antibodies. (B) MDA-MB-231 cells were infected with lentiviruses bearing the indicated shRNAs, and the association of Slug, PRMT5, LSD1 at the promoter of E-cadherin and vimentin was analyzed with the qChIP assay. (C and D) MCF10A cells were infected with lentiviruses carrying the indicated shRNAs together with Slug expression constructs or empty vector. The mRNA or protein level of indicated genes was monitored by qPCR (C) or western blotting (D). (E) MDA-MB-231 cells were infected with lentiviruses carrying the indicated shRNAs, and the association of H4R3me2s, H3R2me2s, H3K9me2 and H3K4me2 at the promoter of E-cadherin and vimentin was analyzed with the qChIP assay. (F and G) MDA-MB-231 cells were infected with lentiviruses carrying shNTC, shLSD1, shPRMT5 and shLSD1 + shPRMT5. The mRNA or protein level of indicated genes was measured by qPCR (F) or western blotting (G). (H and I) SUM159 cells were infected with lentiviruses carrying shNTC, shLSD1, shPRMT5 and shLSD1 + shPRMT5. The mRNA or protein level of indicated genes was measured by qPCR (H) or western blotting (I). (J) MCF10A cells were infected with lentiviruses carrying the indicated shRNAs together with Slug expression constructs or empty vector. The invasiveness of these cells was analyzed with a matrigel-coated chamber invasion assay. (K and L) MDA-MB-231 (K) or SUM159 (L) cells were infected with lentiviruses carrying shNTC, shLSD1, shPRMT5 and shLSD1 + shPRMT5. (M) MDA-MB-231 cells transfected with shSlug and the expression plasmids for PRMT5 or LSD1 for cell invasion assay. For J, K, L and M, representative photomicrographs are shown in the left. The histograms show the mean ± SD of the fold changes of numbers of invasive cells in each of samples, relative to that of control sample from three separate experiments. ***P < 0.001, as determined by Student’s t test. For B, C, E, F and H, the data represent the mean ± SD from three independent experiments (**p < 0.01, ***p < 0.001, Student’s t-test)