Extended Data Fig. 3: PI3K inhibition induces ROS in breast cancer cells.

Relative viability of HCC1954 cells treated with lapatinib and 5 mM NAC for 72 hours. Data are shown as the mean ± SEM for n = 3 biologically independent replicates. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test). b, DCFDA staining showing ROS levels in BT474 and SKBR3 cells treated with lapatinib or 100 μM dehydroepiandrosterone (DHEA) for 48 hours. BT474 cells were treated with 100 μM tert-butyl hydrogen peroxide (TBHP) for 12 hours as a positive control. Data are shown as the mean of n = 2 biologically independent replicates and are representative of 2 biologically independent experiments. c, DCFDA staining showing ROS levels in BT474 cells treated with lapatinib and 50 μM 6-aminonicotinamide (6-An) for 48 hours. Data are shown as the mean of n = 2 biologically independent replicates and are representative of 2 biologically independent experiments. d, DCFDA staining showing ROS levels in mammospheres treated with 50 μM 6-An or 100 μM TBHP for 24 hours. Data are shown as the mean of n = 2 biologically independent replicates and are representative of 2 biologically independent experiments. e, Relative viability of mammospheres cultured with or without dox in the presence or absence of 100 μM etomoxir (eto) for 7 days. Data are shown as the mean ± SEM for n = 3 biologically independent replicates. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. f, qRT-PCR analysis of Cpt1a, Cpt1b, and CD36 expression in 2 independent mammosphere cultures grown in the presence of dox or without dox for 4 days. Data are shown as the mean ± SEM for n = 3 biologically independent replicates. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. g, Boron-dipyrromethene (BODIPY) staining showing lipid droplets (green) in mammospheres cultured in the presence of dox. Scale bar, 25 μm. h, DCFDA staining showing ROS levels in T47D, BT474, MDA-MB-231, and SKBR3 cells treated with 500 nM BKM120 for 48 hours. Data are representative of n = 3 biologically independent replicates for a single experiment. Significance was determined by two-sided Student’s t test. i, DCFDA staining showing ROS levels in MDA-MB-231 cells treated with 500 nM JQ1 for 48 hours. Data are representative of n = 3 biologically independent replicates for a single experiment. Significance was determined by two-sided Student’s t test.