P pi3k 17366

VPA (P4543), melatonin (M5250), and DCFH-DA (D6883) were ordered from Sigma (St Louis, USA). Primary antibodies against src (#2109), p-src (#12432), ERK (#68303), p-ERK (#4370), PI3K (#4249), and p-PI3K (#17366) were purchased from Cell Signaling Technology (Danvers, USA); antibodies against PH3 (ab177218), Ki67 (ab16667) and BCL-2 (ab182858) were ordered from Abcam (Cambridge, USA); and GAPDH (60004-1-Ig) was purchased from Proteintech Group (Wuhan, China). FAST DiO (D3898), DMEM (11960044) and fetal bovine serum (10099141) were purchased from Thermo Fisher Scientific (Waltham, USA). The ROS Assay Kit was purchased from Beyotime (Shanghai, China).

Whole-cell lysates were lysed from cell lines using RIPA buffer (Thermo Scientific, USA). The concentration of lysates was quantified using the Bicinchoninic Acid protein assay kit (Thermo Fisher Scientific, Cambridge, MA, USA). An equal amount of proteins (50 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto the polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% (w/v) skim milk, the blotting PVDF membranes were separately incubated with specific primary antibodies against Bcl-2, 4223; Bax, 14,796; p-PI3K, 17,366; (1:1000, Cell Signaling Technology, USA), PI3K, ab32089; AKT, ab8805; GAPDH, ab181606 (1/1000, Abcam, USA), and p-AKT, SAB4301414 (1:1000, Sigma-Aldrich, USA) overnight at 4 °C. Subsequently, PVDF membranes were blocked with goat anti-rabbit IgG HRP-linked secondary antibody (1:5000, Abcam) at room temperature for 1 h. The signal bands were measured with an Enhanced Chemiluminescence Kit (Thermo Fisher Scientific, Cambridge, MA, USA) and the band intensity was analyzed by Quantity One software (BioRad, Hercules, CA, USA).