(A,B) Effect of Dox depletion for 3 days on levels of Gli2 (A) and Gli1 (B) mRNA as measured by qRT-PCR in the indicated iKRAS cell lines, (n = 3). (C) Immunoblot shows expression of GLI2 and KRAS in iKRAS2 cells + Dox and – Dox for 3 and 5 days. (D,E) Effect of shRNA-mediated knockdown of KRAS on GLI2, GLI1, ZEB1 mRNA levels (D) and basal-like gene expression (E) as measured by qRT-PCR in YAPC cells. (n = 3). (F) Fold change in the basal-like gene signature in iKRAS4 cells following Dox removal (red plot) for 3 days or Dox removal in conjunction with 5 μM GANT61 treatment (grey plot). qRT-PCR measurement of the indicated genes in both conditions are normalized to the +Dox control condition (black plot) (n = 3). (G) Colony-forming ability of iKRAS4 cells stably expressing empty vector (EV) or Flag-tagged mGLI2 (GLI2) grown in the presence (top) or absence (bottom) of Dox for 6 days, followed by staining with crystal violet. Graph shows the fold change in growth relative to the EV +Dox setting. Results shown are representative of n = 3 experiments. (H,I) Images (H) show the effect of stable expression of EV (top) or Flag tagged GLI2 (bottom) on YAPC sphere formation 5 days post shRNA-mediated knockdown of KRAS compared to control cells (shGFP). Quantification of sphere area for each condition is shown (I). Number of spheres measured per condition is indicated in parenthesis. Scale bar, 200 μm. (J) Immunoblots show expression of the indicated proteins in iKRAS (black) and Escaper (red) cell lines. Numerical values indicate levels of GLI2 and YAP1 normalized to Tubulin. Escaper cell lines denoted with ‘Y’ harbor genomic amplification of Yap1. (K–M) Images (K) show the effect of shRNA-mediated knockdown of Gli2 using two separate hairpins on iKRAS1 sphere formation when grown in the presence (top) or absence (bottom) of Dox for 18 days. Quantification of sphere area in the presence (L) and absence (M) of Dox is shown. Number of spheres measured per condition is indicated in parenthesis. Arrowheads indicate growth of KRASG12D independent spheroids. Scale bar, 100 μm. p-Values were calculated by two-tailed unpaired t test. n.s. = not significant, *p<0.05; **p<0.01; ***p<0.001.