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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 2. The noncanonical pathway contributes marginally to SMC3-induced NF-κB activation and is dispensable for SMC3-induced TNF secretion.

Figure 2

A, H23, HepG2, Huh-7, and MCF-7 cells were cotransfected with p5×κB-Luc and pRSV-LacZ. Twenty-four hours after transfection the cells were treated with SMC3 (50 nM for H23 and 100 nM for the rest cells) for 24 h or left untreated. Luciferase activity was detected and normalized to β-galactosidase activity. Data shown are the mean ± SD. B, Upper panel, H23 cells were treated with SMC3 (50 nM) for the indicated times. Bcl-XL and MnSOD proteins were detected by Western blot. β-Tubulin was detected as an input control. Lower panel, Bcl-XL and MnSOD RNAs were detected by RT-PCR. β-actin was detected as an input control. C, Upper panel, H23 cells were treated with SMC3 (50nM) for indicated times. NF-κB p100 and p52 were detected by Western blot. For detecting the weak p52 signal, a long-time exposure was used (middle panel). β-Tubulin was detected as an input control. D, H23 cells were mock transfected or transfected with 5 nM of RelB-siRNA or negative control siRNA. Forty-eight h after transfection the cells were cotransfected with p5×κB-Luc and pRSV-LacZ. Twenty-four hours post-transfection the cells were treated with SMC3(50 nM) for 24 h or left untreated. Luciferase activity was detected and normalized to β-galactosidase activity. Insert, knockdown of RelB in H23 cells detected by Western blot. E, H23 cells were mock transfected or transfected with 5 nM of RelB- or negative control siRNA. Forty-eight h after transfection the cell were treated with SMC3 for 24 hours or left untreated; the TNF concentrations in the cell culture media were measured.