(A) Heatmap showing the expression of mesenchymal (FN1, VIM, ZEB1) and epithelial (CDH1, KRT14) markers in HME cells expressing the indicated candidate ORFs, normalized to a negative control (EGFP) and quantified by qPCR. n = 3. Bar graphs and statistical significance for each gene are shown in Figure 2—figure supplement 1B. (B) Levels of EMT marker expression in HME cells expressing the indicated candidate ORFs quantified by immunoblotting. (C) The rate of mammosphere formation in HMLE cells that express the indicated ORFs. The number of mammosphere per 1000 cells plated was counted. n = 5, ***, p<0.001; **, p<0.01; *, p<0.05. Representative images are shown in Figure 2—figure supplement 1C. (D) In vivo tumor formation assay. HMLER cells expressing the corresponding ORFs were injected subcutaneously into immuno-compromised mice and tumor growth was monitored for 15 weeks. Shown are an image of tumor sizes (left) and quantification of tumor weights (right) (individual tumor growth curves are shown in Figure 2—figure supplement 1E). n = 9, **, p<0.01; *, p<0.05, Student’s two-tailed t-test. (E) The percentage of CD44-high cells when SNAI1 is overexpressed along with shRNAs targeting candidate RNA-binding proteins or LacZ as a negative control. The red bar indicates a positive control shRNA against SNAI1 and the orange bars indicate shRNAs targeting QKI and RBFOX1. The knockdown efficiency is shown in Figure 2—figure supplement 3D. n = 4, ***, p<0.001; **, p<0.01; *, p<0.05, Student’s two-tailed t-test. (F–I) The effect of CRISPR/Cas9-targeting of endogenous QKI or RBFOX1 in HMLE cells ectopically expressing SNAI1 on: (F) the percentage of CD44-high cells, (G) EMT marker expression (VIM: mesenchymal marker, CDH1: epithelial marker) quantified by qPCR, (H) EMT marker expression quantified by immunoblotting and (I) mammosphere formation. Also shown is the efficiency of gene knockout as quantified by immunoblotting. (B) n = 4, (C) n = 4, (E) n = 3, ***, p<0.001; **, p<0.01; *, p<0.05, Student’s two-tailed t-test. (J) Summary of effects of ORFs on EMT markers and phenotypes.