Cyclin d1 e3p5s

For Western blot analysis, pelleted cells were lysed, protein concentration was determined, and whole cell extracts were used for SDS-Page and semi-dry Western blotting, with subsequent detection and quantification as described before in Ref. [2 (link)] Target detection was performed with antibodies against SGPL1 (HPA021125; Atlas Antibodies, Bromma, Sweden,), panAKT (C67E7), Phospho-Ser472-AKT (D9E), Phospho-Thr308-AKT (D25E6), FOXO3 (D19A7), p27kip1a (D69C12), Cyclin D1 (E3P5S) from Cell Signaling, CDK2 (E304, Abcam), and β-Actin (A5441, Sigma-Aldrich) and according secondary antibodies, anti-rabbit IgG or anti-mouse IgG (GE Healthcare, Little Chalfont, UK).

Compounds 1, 2, and 3 were synthesized according to the procedure developed by our group [12 (link)]. They were dissolved in dimethyl sulfoxide (DMSO), the concentration of which never exceeded 0.1% (v/v); 50 mM of stock solution was stored at −20 °C. The Cell counting Kit-8 (CCK-8), Hoechst 33258, JC-1, and the ROS assay kit were purchased from Beyotime Institute of Biotechnology Company (Shanghai, China). The annexin V-FITC and propidium iodide (PI) kit was obtained from BD Pharmingen (BD, San Diego, CA, USA). The primary antibodies used are as follows: CDK4 (D9G3E, Cell Signaling Technology, Danvers, MA, USA), Cyclin D1 (E3P5S, Cell Signaling Technology), Caspase-3 and cleaved Caspase-3 (D3R6Y, Cell Signaling Technology), PARP (46D11, Cell Signaling Technology), Cytochrome c (AF0146, Affinity, Changzhou, China), Caspase-9 (AF6348, Affinity), cleaved Caspase-9 (D8I9E, Cell Signaling Technology), BAX (D2E11, Cell Signaling Technology), Bcl-2 (D17C4, Cell Signaling Technology), Bad (D24A9, Cell Signaling Technology), and β-actin (AF0003, Beyotime Biotechnology, Shanghai, China). All other chemicals used are commercially available and reagent grade.