Alpelisib byl719

Lapatinib (Selleck Chemicals, #S2111), alpelisib (BYL719, Selleck Chemicals, #S2814) and cobimetinib (GDC‐0973, Selleck Chemicals, #S8041) were dissolved in DMSO (Sigma, #D2650) for in vitro studies and in 0.5% CMC‐Na (Selleck Chemicals, #S6703) for in vivo studies. The primary antibodies used for western blotting included those against p‐HER2‐Tyr1221/1222 (Abcam, #ab131102, 1:500), HER2 (CST, #2242, 1:1000), pIGF‐1R (CST, #3021, 1:500), IGF‐1R (CST, #9750, 1:500), p‐AKT‐Ser473 (CST, #4060, 1:1000), p‐AKT‐Thr308 (CST, #13038, 1:1000), AKT (CST, #4691, 1:1000), p‐ERK‐Tyr202/204 (CST, #4370, 1:1000), ERK (CST, #4695, 1:1000), p‐MEK‐Ser217/221 (CST, #9154, 1:1000), MEK (CST, #9126, 1:1000), p‐S6‐Ser240/244 (CST, #5364, 1:1000), S6 (CST, #2217, 1:1000), HA‐tag (CST, #3724, 1:1000), p110α (CST, #4249, 1:1000) and vinculin (CST, #13901, 1:2000), and HRP‐conjugated goat‐anti‐rabbit secondary antibody (Invitrogen, #31460, 1:5000). Exposure time of each band depends on the saturation degree of chemiluminescent, generally no more than 1 min.

To test the effect of drug treatment on protein and phosphoprotein levels, cell lines were dosed with drugs focused on the PI3K pathway including buparlisib (BKM-120, Selleck Chemicals), alpelisib (BYL-719, Selleck chemicals), IPI-549 (Selleck Chemicals), sirolimus (rapamycin, Selleck Chemicals), and ipatasertib (GDC-0068, Selleck Chemicals). To determine dosing for imaging-based experiments, all drugs were screened against each cell line to determine IC₅₀ values via conventional cell viability assay (MTT). Treatments were administered at 80% of the IC₅₀ dosing overnight prior to fixation for cell line treatments (Fig. 5b, Supplementary Fig. 10).