Sb216763

H460 cells were transfected with GSK-3β-WT, GSK-3β-S9A, GSK-3β-K85R, or negative control plasmids for 48 h. A549 cells were treated with the GSK-3 inhibitor SB216763 (Cell Signaling Technology, Danvers, MA, USA) for 24 h, and H460 cells were treated with rapamycin (KeyGEN Biotech, Nanjing, China) and 3-methyladenine (3-MA, KeyGEN Biotech) for 24 h. Cells were then irradiated with 2-Gy X-rays, transferred to three 6-cm cell culture dishes (1000 cells per dish) and incubated for 2 weeks. Then, the plates were washed with PBS and stained with Giemsa solution. Colonies with more than 50 cells were counted under a microscope. All experiments were performed in triplicate.

Methazolamide (MTZ, N-[5-(aminosulfonyl)-3-methyl-1,3,4-thiadiazol-2(3H)-ylidene]-acetamide), melatonin (MEL, N-acetyl-5-methoxy tryptamine), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), and sulforaphane (SFN, 1-isothiocyanato-4-(methylsulfinyl)-butane) were procured from Sigma. The PI3K inhibitors Wortmannin and LY294002 were from Millipore-Sigma (Burlington, MA) and Cell Signaling (Danvers, MA), respectively. The GSK-3 inhibitor SB216763 was purchased from Cell Signaling.

Male Sprague-Dawley (SD) rats (2 months old, 250-300 g) were provided by Experimental Animal Center of Southern Medical University, Guangdong, China. Efforts were made to minimize the number used and suffering of animals. All animals were housed under climate-controlled condition, and free to get standard rodent diet and tap water. Dexmedetomidine was from Jiangsu Heng Rui Medicine (Jiangsu, China). The yohimbine hydrochloride, 2,3,5-triphenyl-tetrazolium chloride (TTC), 4′,6-diamidino-2-phenylindole (DAPI), as well as mouse anti-Cx43 and mouse anti-β-tubulin antibodies were from Sigma (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody came from Jackson ImmunoResearch Laboratories (West Grove, Pennsylvania, USA). Rabbit polyclonal anti-Cx43 antibody, LY294002, SB216763, anti-mouse Alexa 488, and anti-rabbit Alexa 555 were from Cell Signaling Technology (Beverly, MA, USA). The mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody was from Abcam (Cambridge, UK). Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS) and dulbecco’s modified eagle medium (DMEM) were from Gibco (Auckland, New Zealand). 5% goat serum was from Solarbio (Beijing, China).