Idin-horseradish peroxidase complex (SABC) for 30 min at room temperature. After treated
Idin-horseradish peroxidase complex (SABC) for 30 min at room temperature. After treated with diaminobenzidine (DAB) Kit (ZhongshanGoldenbridge, Beijing, China), the stained slides were counterstained with hematoxylin and mounted. Negative control was carried out with the primary antibody replaced PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26509685 by PBS. IHC staining was scored by the proportion of positive tumor cells. Five microscopic fields with the highest immunoreactivity at ?00 magnification were evaluated by two independent observers who were blinded to the patients’ clinical data. Cases with at least 10 of tumor cells with Klotho staining were considered as positive. Fresh mice subcutaneous tumors were fixed in 4 paraformaldehyde (PFA) and embedded with paraffin for histological examinations. Sections with 4-m thickness were cut and stained with hematoxylin-eosin (H E).Western blottingwere performed in triplicate with GAPDH or -actin (Zhongshan Goldenbridge, Beijing, China) as endogenous control.Cell proliferation assayCell proliferation was assessed by performing triplicate assays with the Cell Counting Kit-8 (CCK-8) assay (Enogene, Nanjing, China). DLBCL cells with designed treatment were seeded in 96-well plates at a density of 5000 cells/well for 48 or 24 96 h later. Thereafter, the cells were incubated with 10 l/well CCK-8 for 4 h according to the manufacturer’s proposal. Cell proliferation was detected by light absorption at 450 nm by Multiskan GO Microplate Reader (Thermo Scientific, Rockford, IL, USA).Flow cytometry analysisCells were lysed in radio-immunoprecipitation assay buffer (Shenergy Biocolor, Shanghai, China) together with 1?phosphatase inhibitor cocktail (PhosSTOP; Roche, Mannheim, Germany). The BCA assay (Shenergy Biocolor, Shanghai, China) was performed to detect protein concentration. Protein extracts (30 g) were then electrophoresed on SDS-polyacrylamide gels and blotted from the gel onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were incubated with the blocking solution (5 skim milk in Tris-buffered saline containing 0.05 Tween-20) for 1 h at room temperature and then immunoblotted with the indicated antibodies (1:1000 dilution) overnight at 4 ?C. After which, the membranes were washed with TBS-T and then probed with the HRP-conjugated secondary antibodies (Zhongshan Goldenbridge, Beijing, China) and the electro-chemi-luminescence kit (Millipore, Billerica, MA, USA). Chemiluminescent signals were detected using the Amersham Imager 600 imaging system (General Electric, USA). ImageJ software (ImageJ, NIH) was used to quantify the protein bands normalized to control. Primary antibodies used were Klotho (Abcam, SIS3MedChemExpress SIS3 Cambridge, MA, USA), phospho-IGF-1R (Tyr1135/1136), IGF-1R, phospho-AKT(Ser473), total pan-AKT, Mcl-1, Caspase-3, diphosphorylated and total ERK1/2 (Cell Signaling Technologies, Beverly, MA, USA), -actin, and GAPDH (Zhongshan Goldenbridge, Beijing, China). The experimentsApoptosis of transfected DLBCL cells were detected by Annexin V-PE/7-aminoactinomycin (7AAD) (BD Biosciences, Bedford, MA, USA) assay according to the manufacturers’ instructions. DLBCL cells with designed treatments were harvested and washed twice with icecold PBS and incubated in 1?binding buffer (containing 5 l Annexin V-PE and 5 l 7AAD). After incubation in the dark for 15 min, cells were subjected to the flow cytometry. At least 10,000 events per sample were acquired. Cells were discriminated into viable cells (AnnexinV-PE-/7AAD-), dead cells.