EGFR inhibitor was from Roche. EGFR, ErbB2, ErbB3 and ErbB4 antibodies for immunofluorescent staining were purchased from Abcam. Cell selleck products IQ live cell imaging platform was manufactured by Chip mantech and equipped in Center for Biomedical Research, Zhongshan Hospital, Fudan Uni versity, Shanghai, China. Measurement of gene expression Total RNA was isolated using a guanidinium isothiocyan atechloroform based technique and measured with OD 260 nm. RNA was subse quently Inhibitors,Modulators,Libraries reversed and transcribed to cDNA with the Super Script First strand Synthesis System. Quantitative RT PCR was carried out using an ABI 7000 PCR instrument with the two stage program parameters, as follows 1 min at 95 C, and then 40 cycles of 5 s at 95 C and 30s at 60 C.
Inhibitors,Modulators,Libraries The sequences of the primer sets used for this analysis are as fol lows BTC, Specificity of the produced amplification product was confirmed by examination of dissociation re action plots. Each sample was tested in triplicate with quantitative RT PCR, and each group had six wells. Production of BTC and CXCL8 A549 cells were cultured in 24 well cell culture micro plates at 1105 cellswell for 24 h and then treated with lipopolysaccharide at concentrations of 0. 01, 0. 1, and 1 ugml for an additional 24 h, respectively, to study LPS induced productions of BTC or CXCL8. Cells were pre incubated with an anti human BTC neutralizing antibody at concentrations of 1, 10, 100 ngml or IgG as non specific control 2 h before LPS stimulation to study the role of BTC in LPS induced CXCL8 production. Cells Inhibitors,Modulators,Libraries were treated with BTC at 0.
1 ugml or vehicle and pretreated with BEZ235, GDC0941, SHBM1009, Inhibitors,Modulators,Libraries Erlotinib, or PD98059 at 0. 1, 1, or 10 uM, respectively, for 24 h to investigate the involvement of various sig nal pathways. Each experiment was done in six repli cate Inhibitors,Modulators,Libraries wells for each drug concentration and each time point. Levels of BTC and CXCL8 proteins in super natant were measured by ELISA at the absorbanceof 450 nm. Expression of receptors A549 were fixed with 4% paraformaldehyde, washed thrice, permeabilized with 0. 1% Triton X 100, and blocked with 10% goat serum. Cells were incubated overnight with mouse monoclonal antibodies against EGFR, ErbB2, ErbB3, or ErbB4, respectively, and then strained with FITC conjugated anti mouse IgG antibody. The counterstaining was performed with DAPI and cells were examined under immunofluorescence microscope.
Measurements of apoptosis Apoptosis was analyzed by FACS as previously described. Lung cancer cells A549 were treated with TNF TNF-�� inhibitor at 20 ngmlCHX at 2. 5 umoll for 24 h in the presence or ab sence of BTC at 0. 01, 0. 1, 1 ugml, respectively. Cells were then harvested and washed thrice, resuspended in pre diluted binding buffer, and stained with Annexin V FITC for 20 min. Cell apoptosis was analyzed by flow cytometry using Cell Quest Software.
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