The lower chambers contained complete medium with 0, 2, 4 and 8 M rottlerin. human GC cell lines. Furthermore, the molecular mechanism underlying the antitumor activity of rottlerin through activation of autophagy in GC cells was investigated, and the results indicate that rottlerin-induced autophagy may promote anticancer activity through cancer cell apoptosis. Materials and methods Cell culture and reagents The SGC-7901 and MGC-803 human GC cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% streptomycin and penicillin in a humidified incubator at 5% CO2 and 37C. Rabbit primary antibodies against S-phase kinase-associated protein 2 (Skp2; cat. no. ab183039), mechanistic target of rapamycin kinase (mTOR; cat. no. ab32028), microtubule-associated protein 1 light chain 3 (LC3)-II (cat. no. ab51520), caspase-3 (cat. no. ab13847), cleaved-caspase-3 (cat. no. ab2302), poly(ADP ribose) polymerase (PARP; cat. no. ab32138) and cleaved-PARP (cat. Alosetron (Hydrochloride(1:X)) no. ab32064) were purchased from Abcam (Cambridge, UK). Rabbit primary antibodies against -actin (cat. no. 4970) were purchased from Cell Signaling Alosetron (Hydrochloride(1:X)) Technology, Inc. (Danvers, MA, USA). Secondary antibody (goat anti-rabbit; cat. no. sc2004) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rottlerin and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rottlerin was dissolved in DMSO to generate a 10 Alosetron (Hydrochloride(1:X)) mM stock solution. Cells cultured with only 0.1% DMSO served as the control group. Cell proliferation assay Cell proliferation was measured with a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). SGC-7901 and MGC-803 cells were seeded in 96-well plates at Alosetron (Hydrochloride(1:X)) a density of 2,000 cells/well and incubated in a humid environment at 5% CO2 and 37C for 4 h. Subsequently, the cells were exposed to 0, 1, 2, 4, 8 and 16 M rottlerin for 12, 24, 48 and 72 h. CCK-8 reagent was then added and incubated for 30 min at 37C. Absorbance of the colored formazan product, formed by mitochondrial dehydrogenases, was measured at a wavelength of 450 nm. Colony formation assay SGC-7901 and MGC-803 cells were cultured in a 6-well plate at a density of Alosetron (Hydrochloride(1:X)) 500 cells/well with 0, 2, 4 and 8 M rottlerin at 37C for 2 weeks. Cells treated with rottlerin-free medium served as the control group. After 2 weeks, the cells were fixed in 4% methanol for 15 min at room temperature. Cells were then stained with 0.1% crystal violet for 5 min at room temperature and imaged using a light microscope (Olympus Corporation, Tokyo, Japan) at 40 magnification. Cell cycle assay SGC-7901 and MGC-803 cells were seeded at a density of 1106/ml, and then harvested following treatment with 0, 2 4 and 8 M rottlerin at 37C for 24 h. The cells were fixed in 70% ethanol at 4C overnight. The fixed cells were centrifuged at 1,000 g for 15 min at room temperature and washed with cold PBS three times. The cells were incubated with 50 g/ml RNase A at 37C for 30 min. Then cells were incubated with 100 g/ml propidium iodide (PI) in the dark at 4C for 30 min. The DNA content was quantified by FCM (BD CellQuest Pro; BD Biosciences, Franklin Lakes, NJ, USA). The percentages of cells in the G0-G1, S and G2-M phases were compared with the control group. Apoptosis assay SGC-7901 and MGC-803 cells were cultured at 1106/ml in 6-well plates following treatment with 0, 2 4 and 8 M rottlerin at 37C for 24 h. Cells were collected, washed twice with cold PBS and resuspended in 100 l binding buffer containing 5 l fluorescein isothiocyanate-conjugated anti-Annexin V antibody and 5 l PI using a FITC-Annexin V Apoptosis Detection kit (BD Biosciences). Apoptosis was assessed using a FACS Calibur flow cytometer (BD CellQuest Pro; BD Biosciences). The percentages of apoptotic cells were compared with the control group. Cell migration and invasion assays SGC-7901 and MGC-803 cells were cultured at 1106/ml Rabbit Polyclonal to CSF2RA in 6-well plates. Migration was assessed.