Extended Data Fig. 3: PRC2 and KMT2D-COMPASS regulate EMP.
a, Sanger sequencing demonstrate complete knock-out of ASH2L, EED and KMT2D genes in the corresponding clonal cells. b, Percentage of CD44hi mesenchymal population in C1 cells transduced with sgRNAs targeting SETD1A, SETD1B, KMT2A, KMT2B, KMT2C and KMT2D respectively. n=3. ***, p<0.001. Statistical analysis was performed using one-way ANOVA followed by Dunnett multiple-comparison analysis. Data are presented as mean ± SEM. c, Flow cytometry analysis shows the CD44 and CD104 cell-surface staining of sorted epithelial subpopulation from C1-sgEED and C1-sgKMT2D cells (left) and the quantification of CD44hi mesenchymal population in different culture conditions (right). Cells were cultured in control (DMSO) or SB-431542 (5 𝜇M) treated condition in vitro for 5 days. n=3. **, p = 0.001 (C1-sgEED-Epi), 0.007 (C1-sgKMT2D-Epi). Statistical analysis was performed using unpaired two-tailed Student t-tests. Data are presented as mean ± SEM. d, Flow cytometry of the CD44 cell-surface staining of C3-sgControl, C3-sgEED and C3-sgKMT2D cells at the population level. e, Flow cytometry of the EpCAM cell-surface staining of HCC827-sgControl, HCC827-sgEED and HCC827-sgKMT2D cells at the population level. f. Flow cytometry of cell-surface EpCAM in SUM149D2-sgControl, SUM149D2-sgEED and SUM149D2-sgKMT2D cells at the population level. g, Immortalized but not transformed HMLE epithelial cells contain convertible (nrc-4) and non-convertible (nrc-1) single cell clones. RAS transformation promotes EMT in convertible clone but not in non-convertible clone. h, Immunoblot of E-cadherin, N-cadherin, and ZEB1 in representative HMLE clones before and after RAS oncogene transformation. GAPDH as loading control. n = 2 biologically independent experiments. i, Flow cytometry of the CD44 and CD104 cell-surface staining of HMLE-nrc-1-sgControl, HMLE-nrc-1-sgEED and HMLE-nrc-1-sgKMT2D cells in control or TGF-β treated (2 ng/ml) conditions for 7 days. HMLE-nrc-1 is a clonal cell population generated from HMLE that stably reside in an epithelial state. Numerical source data are provided.
