Figure 3

ASPP2 inhibits NF-κB/p65-dependent transactivation of BECN1. (a) Various truncated BECN1 promoters were generated by PCR and inserted into reporter vector pGL3.1 (upper). These constructs were co-transfected with an internal control vector into HCC-LM3 cells with overexpression or silencing of ASPP2. The cells were collected for analysis of luciferase activities (middle). The JASPAR database was queried for consensus binding sites of known transcription factors for the sequence nt −305 to −296 and nt +22 to +31 of the BECN1 promoter (lower). (b) HCC-LM3 cells transfected with vector or pASPP2 were transfected with BECN1 (−625/+155) and (−156/+155)-luc or the promoter containing p65/RelA consensus binding site mutation (cggggtttca→aaattgaaga, BECN1-625/ΔRelA) and (gggaagtcgc→aagatgaagc, BECN1-156/ΔRelA), and luciferase activities were measured at 48 h posttransfection followed by 6 h EBSS treatment. (c) HCC-LM3 cells were co-transfected with BECN1 (−156/+155)-luc and increasing amounts of ASPP2-expressing plasmids (0.5, 1.0, and 1.5 versus control). (d) HepG2 cell infected with LV-shNon or LV-shASPP2 were transfected with BECN1 (−156/+155)-luc, and Huh7 cell lines were co-transfected with BECN1 (−156/+155)-luc and pASPP2 (right). (e) HCC-LM3 cells were co-transfected with vector or pASPP2 and NF-κB-driven luciferase construct (left). HCC-LM3 cells transfected as indicated, the nuclear and cytoplasm extracts were immunoblotted with the indicated antibodies (right). (f) HCC-LM3 cells transfected with pASPP2-V5 were stained with anti-p65/RelA (green) and anti-V5 (red) antibodies and imaged by IF. Scale bars: 30 μm (left). The ASPP2-p65/RelA and ASPP2-IκBα conjugates were immunoprecipitated with anti-ASPP2 and analyzed by western blotting (right). (g) HCC-LM3 cells transfected with pASPP2-V5 were stained with anti-V5 (red) and anti-IκBα (green) antibodies. Scale bars: 30 μm (left). Cell lysates were subjected to western blotting (right). (h) The IκBα-p65/RelA complex was immunoprecipitated with anti-p65/RelA and analyzed by western blotting. (i) Chromatin-immunoprecipitation analysis was performed on HepG2 and Huh7 cell lysates using antibodies against p65/RelA. Data are shown as the means±S.D. from triplicate experiments. (*P<0.05; **P<0.01)