(A and B) ETC-159 treatment of HPAF-II cells increases abundance of EGFR on the cell surface. HPAF-II cells were treated with 100 nM ETC-159 for 72 hours. (A) Cells were stained with Alexa Fluor 488–conjugated anti-EGFR antibody and analyzed by flow cytometry. Each histogram represents ~50,000 cells. Data are representative of 3 independent experiments (P = 0.009). (B) Endogenous EGFR levels on non-permeabilized cells were assessed by indirect immunofluorescence microscopy. Scale bars: 20 μm. (C and D) Partial knockdown of RAB11B or EHD1 blunts the ETC-159–induced EGFR increase on the surface of HPAF-II cells. HPAF-II cells were transfected with 2 independent siRNAs (si6 and si7) against RAB11B (C) or EHD1 (D) for 24 hours, followed by treatment with ETC-159 for 72 hours. EGFR levels on the cell surface were assessed by flow cytometry. Each histogram represents ~50,000 cells from 1 replicate. Data are representative of 3 independent experiments. Average median fluorescence intensity (MFI) of the technical replicates is shown. Av MFI, average MFI of the technical replicates from the same experiment. (E) EPHB2 and EPHB4 levels are increased by Wnt inhibition. HPAF-II cells were treated with 100 nM ETC-159 for 72 hours, and the levels of endogenous EPHB2 and EPHB4 on non-permeabilized cells were assessed by indirect immunofluorescence microscopy. Scale bars: 20 μm. (F–I) Wnt inhibition increases activation of EPHA2 and EGFR receptor tyrosine kinases and protein abundance of multiple receptor tyrosine kinases in HPAF-II xenografts and pancreatic PDX models. Protein lysates from HPAF-II orthotopic xenografts or pancreatic PDX from vehicle- or ETC-159–treated mice were analyzed for expression of p-EPHA2 and p-EGFR and abundance of indicated RTKs by Western blots. Each lane represents tumor lysate from an individual mouse. (H) The protein lysates were prepared as a master mix and loaded on independent gels. Only 1 blot was probed for the load control, which is shared with Figure 4F.