Biology Reference
In-Depth Information
19.2.3
Western Blot Analysis
Cisplatin and taurine were treated for 72 h. Cells were lysed in 200 ml RIPA buffer
(50 mM Tris-HCl, pH 8.0, with 150 mM NaCl, 0.1% NP-40, 0.5% sodium deoxy-
cholate, and 0.1% SDS). Equal amount of proteins was separated by 12% SDS-
polyacrylamide gel electrophoresis and electrotransferred nitrocellulose membrane.
The membranes were blocked with Tris-buffered saline (20 mM Tris-HCl and
140 mM NaCl, pH 7.6) containing 0.3% Tween 20 (TBS-T) and 5% nonfat dry milk
at room temperature for 2 h. The membranes were incubated overnight with pri-
mary antibodies at 4°C. After being washed, the membranes were incubated with
horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. All anti-
bodies were purchased from Cell Signaling (Cell Signaling Technology, Inc.,
Danvers, MA, USA). The signal was detected using ECL advanced detection kit
(GE Healthcare Bio-Sciences Corp., NJ, USA).
19.2.4
Microscopic Images of DAPI Staining
Cells were plated at 0.3 × 10
6
cells in 6-well plate. The next day, cells were treated
with various concentrations of taurine and cisplatin for 72 h. After treatments, cells
were washed with cold PBS and then fixed in methanol/DMSO (4:1) solution for
24 h at 4°C. Cells were washed with PBS three times and then stained with DAPI
for 10 min and observed under the fluorescence microscope.
19.2.5
Statistical Analysis
Statistical significance was determined by Tukey multiple comparisons (SigmaStat;
Jandel, San Rafael, CA, USA). Each value was expressed as the mean ± SEM.
Differences were considered statistically significant when the calculated
P
value
was less than 0.05.
19.3
Results
19.3.1
Co-treatment of Cisplatin with Taurine Decreases
Cell Proliferations
Cells were treated with taurine and cisplatin for 48 and 72 h, and cell proliferation
was investigated by MTT assay. In single treatment of taurine, cell proliferation was
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