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. Author manuscript; available in PMC: 2010 Jun 1.
Published in final edited form as: Mol Cancer Ther. 2009 Jun 9;8(6):1636–1645. doi: 10.1158/1535-7163.MCT-09-0068

Figure 3. The canonical pathway mediates SMC3-induced NF-κB activation but is not required for SMC3-induced TNF secretion.

Figure 3

A, H23 cells were treated with SMC3 (50 nM) for various times as indicated. Phosphorylated- and total IκBα were detected by Western blot. β-Tubulin was detected as an input control. B, H23 cells were mock transfected or transfected with 5 nM of RelA-, IKKβ-, or negative control siRNA. The cells were treated with SMC3 (50 nM) for 24 h and NF-κB activity was analyzed. *p<0.05. C, H23 cells were cotransfected with p5×κB-Luc and pRSV-LacZ. Twenty-four hours post-transfection half of the cells were pretreated with IKK inhibitor II (10 μM) for 1 h followed by SMC3(50 nM) treatment for 24 h or left untreated, Luciferase activity was detected and normalized to β-galactosidase activity. *p<0.05. D, H23, HepG2, Huh-7 and MCF-7 cells were mock transfected or transfected with 5 nM of RelA-, IKKβ-, or negative control siRNA. Forty-eight hours after transfection the cells were cotransfected with p5×κB-Luc and pRSV-LacZ. Twenty-four hours post-transfection the cells were treated with indicated concentration of SMC3 for 24 h or left untreated. The concentrations of TNF in conditioned cell culture media were measured by ELISA. E, The efficiency of knockdown of RelA- and IKKβ-siRNA in H23, HepG2, Huh-7, and MCF-7 cells was detected by Western blot. β-Tubulin was detected as an input control. F, H23, HepG2, Huh-7, and MCF-7 cells were pretreated with IKK inhibitor II (10 nM) for 1 h followed by SMC3 (50 or 100 nM as indicated) treatment for 24 h or left untreated. The concentrations of TNF in conditioned cell culture media were measured by ELISA.