Background The aim of this study was to investigate the effect

Background The aim of this study was to investigate the effect of downregulating Hedgehog pathway by GANT61 on human glioma cells, examine the consequent changes of temozolomide (TMZ)-induced effects and explore the molecular mechanisms. repair enzyme MGMT and the Notch1 pathway increased in the cells treated by TMZ treatment. However, GANT61 could abrogated the protein increasing. Conclusions GANT61 sensitizes glioma cells to TMZ treatment by enhancing DNA damage effect, decreasing MGMT expression and the Notch1 pathway. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0463-3) contains supplementary material, which is available to authorized users. using Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Diego, CA) according to the manufacturers instructions. Annexin V-FITC and propidium iodide (PI) double staining was used to evaluate the percentages of apoptosis. Annexin V? and PI? cells were used as controls. Annexin V+ and PI? 58152-03-7 IC50 cells were designated as apoptotic, and Annexin V+ and PI+ cells as necrotic. Tests were repeated in triplicate. In vitro invasion assays Transwell membranes coated with Matrigel (BD Biosciences, San Jose, CA) were used to assay the invasive ability of glioma cells in vitro. Treated cells were plated at 5??104 58152-03-7 IC50 per well in the upper chamber in serum-free medium. FBS 10% was added to the medium in the lower chamber. After incubation for 24?h, non-invading cells were removed from the top well with a cotton swab, while the bottom cells were fixed with 95% ethanol, stained with 0.1% crystal violet, and photographed in three independent 10 fields for each well. Three independent experiments were conducted and used to calculate fold migration relative to the blank control while the error was calculated as the standard error (SE). Western blot Cell lysates were harvested, equivalent amounts of total protein were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% fat-free milk and 0.1% Tween-20 in tri-buffered saline with Tween (TBST) for 1.5?h at room temperature, membranes were incubated with diluted anti-Gli1, Gli2, E-cadherin, N-cadherin, Vimentin, Fibronectin, MGMT, Notch1, Hes1, H2AX (Ser139) and anti-GAPDH primary antibodies. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies were used, and bound antibodies were detected using the ECL system. Quantitative RT-PCR (qRT-PCR) analysis Total RNA was extracted using Trizol Reagent (Invitrogen, USA) according to the manufacturers instructions. Total cDNA was reversely transcribed from 1?g of total RNA (Perfect Real Time, Takara, Japan). To quantify gene expression, two-step qRT-PCR was performed using a FastStart Universal SYBR Green Master (ROX) by Roche LightCyclerR Real Time System. Expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PCR conditions were hot-start at 95?C (15?s), with annealing and extension at 60?C (60?s) for 40?cycles, followed by a melting curve analysis. All qRT-PCR data were analyzed using the 2-Ct method. And the primers used are shown in Additional file 2. COMET assay The comet assay (Trevigen, Gaithersburg, MD) was performed according to the manufacturers protocol using alkaline conditions. Cell samples were handled under dimmed light to prevent DNA damage from ultraviolet light. Combine cells at 1??105/ml with molten LMAgarose and immediately pipette 50?l onto CometSlide. After placing slides flat at 4?C for 10?min, immerse slides in lysis solution for 60?min and FLJ22263 freshly prepared Alkaline Unwinding Solution, pH?>?13 for 20?min. Electrophoresis was carried out at the rate of 1.0?V/cm for 30?min. The slides were removed from the electrophoresis chamber, washed in deionized water for 5?min and in ice cold 70% ethanol for 5?min. Subsequently, the slides were air-dried, and DNA was stained with 50?l of SYBR Green I dye (Sangon Biotech, 1:10,000 in Tris-EDTA buffer, pH?7.5) 58152-03-7 IC50 for 30?min and immediately analyzed using a fluorescence microscope (Axiovert 200, Carl Zeiss). Data was analyzed using CometScore (TriTek, Sumerduck, VA). Statistical analysis Gene set variation analysis (GSVA) with Gli expression was analyzed by GSVA package [27] of R ( Data are presented as mean??SD. All statistical analyses were performed using SPSS version 13.0 software (Chicago, IL, USA). The Students <0.05 level. Results Gli1 is a prognostic marker in glioma and participates in variety of biological behaviors As shown in Fig.?1a, patients with glioblastoma containing high 58152-03-7 IC50 or low.

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