The results are represented as the mean number of foci or cells per group with lapatinib (50 nM and 100 nM) for 30 min followed by radiation at doses between 2.5C10 Gy. to inhibit tumor cell proliferation and survival . At this time, there have been no previous studies on the effects of the combination of irradiation with lapatinib in treating bladder cancer. Therefore, the aim of this study was to evaluate the effect of lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and HER-2, on the radiosensitivity of murine mouse bladder tumor line-2 (MBT-2) cells and protocol, lapatinib (10 mM) was dissolved in 100% dimethyl sulfoxide (DMSO) and diluted further using culture media. For animal studies, lapatinib was mixed with Tween-80 (0.4%) and methylcellulose (0.5%) in water, and the animals received oral doses of lapatinib. Irradiation of MBT-2 cells Cells of the murine bladder tumor cell line, MBT-2, were exposed to radiation at doses between 2.5C10 Gy using a low energy 6 MV photon beam. Data were obtained at a distance of 100 cm from the source to the surface using an ionization chamber (cylindrical thimble). Clonogenic assay (colony formation assay) To test the effects of lapatinib and irradiation on colony formation, cells were seeded using six-well plates and a cell density of 1105 cells/well. The cells were exposed to different radiation doses, but received pretreatment with lapatinib (200C1,000 nM) for 30 min, with the control cells treated with dimethyl sulfoxide (DMSO). After pre-treatment with lapatinib, and following irradiation, the cells were cultured for a further week. Counting of the cell colonies was done using a light microscope (100 magnification), and the colonies were defined as a group of 50 cells or more. Western immunoblotting using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The cells were washed twice using ice-cold phosphate-buffered saline (PBS), followed by treating with lysis buffer (Sigma, USA). The separation of proteins from cell lysates was done by loading onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The proteins were transferred to polyvinylidene difluoride membrane (PVDF) membranes. The blots were incubated with primary antibodies for 12 h at 4C. Resminostat The bound antibodies were analyzed with selected secondary antibodies. Enhanced chemiluminescence (ECL) (Thermo-Fisher, USA) was performed to identify the bands, according to the manufacturers protocol. The primary antibodies to EGFR, phosphorylated EGFR (p-EGFR), HER-2, and phosphorylated HER-2 (p-HER-2) were obtained from Abcam (Cambridge, MA, USA). The histone variant Resminostat H2AX, phospho-H2AX, Resminostat beta-actin, PARP and cleaved PARP were obtained from Cell Signaling, USA. Cell cycle analysis The cell cycle distribution was done by flow cytometry analysis. Propidium iodide (PI) staining for DNA in cells was analyzed. For the protocol, 106 cells/ml were exposed to lapatinib and irradiation as previously described and were collected after centrifugation. The cells were stained with PI (15 g/ml) in PBS with 5 g/ml DNase-free RNase and Tween-20 (0.5%). The samples were analyzed using an Attune? NxT Acoustic Focusing Cytometer (Thermo Fisher, USA). Immunofluorescence microscopic studies The MBT-2 cells were transferred Resminostat onto coverslips pre-coated with poly-lysine for 12 h to allow the cells to attach to the surface. The cells were exposed to a radiation dose of 2.5 Gy either alone, or in combination with 100 nM of lapatinib. The cells were then incubated for 45 min and were then washed three times with ice-cold PBS, then treated for 30 min with a 4% solution of formaldehyde in PBS for fixation, followed by incubation in 0.5% Triton X-100/PBS for 60 min, 5% bovine serum albumin (BSA) for 60 min, and a final incubation for 2 h with fluorescein isothiocyanate (FITC)-conjugated anti-phospho-Histone -H2AX antibody (Thermo-Fisher, USA) (1: 1500). The cells were washed with PBS and mounted in Vectashield mounting medium containing diamidino-2-phenylindole (Sigma Aldrich USA). A Zeiss LSM 8 microscope was used to examine the -H2AX nuclei at high power, and a mean of at least 120 nuclei was counted. The mean of the -H2AX foci/nuclei indicated the number Resminostat of DNA double-strand breaks. Mouse tumor xenograft model For studies, six-week-old PSFL female C3H/HEN mice were obtained from the Animal Care Center Liaoning Cancer Hospital and Institute. The animals were maintained under controlled conditions in a laminar airflow chamber at room temperature and were fed with a normal pellet diet. The experiments were conducted at the Radiation Oncology Department of Gastrointestinal and Urinary and Musculoskeletal Cancer, Liaoning Cancer Hospital and Institute, Shenyang, China. All the animal experiments received prior approval from the Animal Ethical Committee of Liaoning Cancer Hospital and Institute, Shenyang, China, and the protocols adhered.
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