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. Author manuscript; available in PMC: 2008 Sep 15.
Published in final edited form as: Biochem Pharmacol. 2007 Jun 17;74(6):851–859. doi: 10.1016/j.bcp.2007.06.017

Figure 2. Effect of CtBP1-siRNA on expression of MDR1 mRNA and P-gp.

Figure 2

a. MDR cell lines, NCI/ADR-RES and A2780DX, were treated with 100 nM of a CtBP1-targeted siRNA (CtBP1siRNA), or non-targeting RNA, or mock, which only consisted of oligofectamine and OPTI-MEM® I reduced serum medium. Seventy-two hours later, total RNA was extracted from the cells and the RT reaction was performed. MDR1 and CtBP1 cDNA were amplified by PCR using the MDR1 primer (5′-ATATCAGCAGCCCACATCAT-3′; 5′-GAAGCACTGGGATGTCCGGT-3′) and CtBP1 primer (5′-TACCACACCATCACTCTCAC-3′; 5′-CTCTGGACTCGTGTGCCCTC-3′), respectively. GAPDH was used as an internal control. Aliquots of PCR products were electrophoresed on 1.5% agarose gels, and PCR fragments were visualized by ethidium bromide staining. b. NCI/ADR-RES and A2780DX cells were treated with 100 nM of a CtBP1 siRNA (CtBP1siRNA), or non-targeting RNA, or mock, and cell lysates were prepared from the treated cells. Equal amounts (50 μg proteins) of cell lysates were separated by 6% SDS-polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membrane. The membranes were immunoblotted with monoclonal anti-P-gp, anti-CtBP1, or anti-tubulin antibodies. Detections of proteins were performed using enzyme-linked chemiluminescence. Protein bands were quantified by the Molecular Analyst software. Results are the representative of three similar experiments. c. NCI/ADR-RES and A2780DX cells were treated with 100 nM of CtBP1siRNA-2, and the effect of the siRNA on MDR1 mRNA expression was determined as described above. d. HCT-15 colon carcinoma cells were treated with 100 nM of a CtBP1 siRNA (CtBP1siRNA) or non-targeting RNA, and the effects of the siRNA on MDR1 mRNA and P-gp expression were determined as described above. Results are the representative of two similar experiments.