(A) Stable cell lines generated using CRISPR FOXO3 knockdown have reduced proteins levels of FOXO3. NT and NT_0 are two distinct non-targeting control guides; FOXO3_1 and FOXO3_2 are two distinct guides targeting FOXO3. The experiment and samples from Figs. 4A and 5A are the same, thus, so are the β-actin blots. In Fig. 4A we show part of the blot, while in Fig. 5A we show the full blot. (B-D) FOXO3 knockdown reduces sensitivity to PI3Kα inhibitors alpelisib and taselisib in ER+ breast cancer cell lines MCF7 and T47D. CellTiter-Glo assay was used to measure cell viability before drug treatment and after 6 days of drug treatment. An unpaired, two-sided t-test was used for comparison between groups (panel B). Experiments in panel B single-dose experiments were optimized for the tested PI3Kα inhibitor concentrations: panel B used a larger number of initial cells than panels C and D (5,000 vs 1,000 cells). The relative growth rate is calculated with respect to the growth rate of the DMSO control of each cell line condition. (E) FOXO3 knockdown reduces PI3Kα-inhibitor-induced cell death in MCF7. Cell death was measured using cleaved PARP levels.