Fig. 6

AXL inhibition shrank PC-9 tumors treated with osimertinib in vivo. Stable PC-9 cell lines were generated by the introduction of short hairpin RNAs (shRNA) that mediated inhibition of AXL expression (#37 and #38) and control nontargeting (SCR) shRNA. a The cells were lysed and the indicated proteins were detected by western blotting. b Cells were incubated for the indicated times and their viability assessed using MTT assays. c Cells were incubated with gefitinib or osimertinib at the indicated concentrations for 72 h and the cell viability assessed using MTT assays. d Following the subcutaneous injection of the indicated cells into nude mice, vehicle (control) or osimertinib (2.5 or 5 mg/kg) were administered. Tumor volumes were determined and the results are plotted over time from the start of treatment (mean ± SEM). e Western blotting analysis of the presence of the indicated proteins from the harvested tumors as described for (d). f Quantification of proliferating cells, as determined by their Ki-67-positive proliferation index (percentage of Ki-67-positive cells) as described in the Methods. Columns, mean of five evaluated areas; bars, SD. Comparisons by paired Student’s t tests. g Representative images of PC-9 xenografts containing the indicated shRNAs following immunohistochemical staining with antibodies specific for human Ki-67. Bar, 100 μm