(A) Robust changes in the expression of representative cell-cycle genes over 7 days of PORCN inhibition in HPAF-II orthotopic xenografts. The dotted line is at 1.5-fold (log₂, 0.58). (B) Significant decrease in proliferating cells over time. Ki67-positive cells (left) were quantitated (right) on an entire section that was scanned and analyzed using NIS-Elements software. Two-tailed Mann-Whitney U test was used to calculate significant differences. (C) CCND1 protein changes more robustly compared with mRNA. Blots from Figure 4C were reprobed as indicated. CCND1 mRNA decreases 33% (see A and D), while CCND1 protein decreases approximately 65% after 56 hours treatment in both HPAF-II and CRC PDX models. Each lane is from an independent tumor. Ratios of CCND1 levels to β-actin levels for each lane are indicated. ETC-159 treatment reduces levels of pRb (S780) in both HPAF-II tumors and CRC PDX. Note the actin blots are the same as in Figure 4C. (D) Changes in the expression of CCND1 after 56 hours in CRC PDX model. (E) Representative cell-cycle genes whose expression is influenced by MYC overexpression. Relative expression is plotted (TPM) (left panel) or as log₂ fold change (right panel). (F) Representative cell-cycle genes whose expression is independent of MYC overexpression. Relative expression is plotted as TPM (left panel) or as log₂ fold change (right panel). (G) MYC stabilization only partially rescues proliferation upon PORCN inhibition. Ki-67 staining quantitated as in B. Two-tailed Mann-Whitney U test was used to calculate significant differences.