RNA-Seq analysis to identify gene-expression changes in iPI3K-δ–resistant tumor cells. (A) RNA-Seq analysis of tumors from baseline and first, third, and fourth transfers treated with vehicle or GS-649443. The figure shows selected differentially expressed genes with significant differences in expression levels (fold change > 2 and false discovery rate < 0.05). (B) Protein expression analysis using Simple Western to compare levels of IGF1R, p-AKT, and p-ERK in murine primary tumors from the fourth transfer, always treated with vehicle (iPI3K-δ sensitive) or GS-649443 (iPI3K-δ resistant). (C) Fold change in Igf1r expression measured ex vivo by RT-qPCR in tumors from the first to fourth transfers. The blue arrows represent the tumors that were transferred to the subsequent rounds. Mann-Whitney U tests were performed to analyze differences in Igf1r expression between the GS-649443 treatments (green vs red, second transfer, P = .0095; third transfer, P = .0061; fourth transfer, P = .007. (D) Protein expression of PI3K isoforms analyzed using Simple Western in iPI3K-δ–sensitive (fourth-transfer tumors treated with vehicle in all rounds) and iPI3K-δ–resistant (fourth-transfer tumors treated with GS-649443 in all rounds) tumor cells. **P ≤ .01.