FIG. 2.

ING4 reduces COX-2 and MMP-9 expression and binds NF-κB p65. (A) U251-TR/F-ING4 cells were grown in the absence (−) or presence (+) of Tet or PMA for 24 h, and total protein was analyzed by immunoblotting assays with antibodies specific for ING4 and actin. (B) The levels of endogenous ING4 in U87-MG and U118-MG cell lines were compared to exogenous F-ING4 levels in U251-TR/F-ING4 clones 7 and 210 grown in the presence of Tet (+) using immunoblot analyses. Forty micrograms of total protein from each sample was evaluated using two different ING4 antibodies (r = rabbit; g = goat). Clone 210 was chosen for studies outlined herein. (C) U251-TR/F-ING4 cells were grown in the absence or presence of Tet for 24 h and in the absence or presence of PMA for 4 h (COX-2) or 24 h (MMP-9). Total RNA was purified and analyzed by RT-PCR using primers specific for ING4, COX-2, MMP-9, and GAPDH. Densitometry was performed, and the levels of COX-2 and MMP-9 in the absence of Tet and PMA were set at 1, and the (fold) change in induction is shown. Data shown are representative of three experiments. (D) U251-MG (U251) and U251-TR/sh-p65 cells, which inducibly express shRNA specific for p65, were grown in the absence or presence of Tet for 48 h and then in the absence or presence of PMA for 24 h. Forty micrograms of total protein were analyzed by immunoblotting analyses using the antibodies specified. (E) Recombinant F-p65 and F-ING4 proteins were produced by in vitro transcription and translation. Equal volumes of reaction mixtures containing no template (−), F-p65, or F-ING4 were mixed and then immunoprecipitated using normal rabbit serum (IgG), anti-ING4, or anti-p65 antibodies. Immunoprecipitated proteins were analyzed by immunoblotting with anti-Flag antibodies. Arrowheads identify F-ING4 and F-p65 proteins. (F) U251-MG cells were transfected with a plasmid carrying p65 and either an empty plasmid (−) or a plasmid encoding F-ING4 (+). Cells were grown for 24 h, and total protein was collected and immunoprecipitated (IP) with a p65 antibody and analyzed by immunoblotting (IB) with an antibody specific for the Flag epitope. Input samples were analyzed with anti-p65 and Flag antibodies. The location of F-ING4 protein is indicated with an *. (G) U251-MG cells were grown on coverslips and transfected with a plasmid carrying either ING4-GFP or GFP alone. Cells were grown in the absence or presence of PMA for 1 h and then stained for total p65 (p65) or p-p65 (S276). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) and imaged using indirect immunofluorescent microscopy. The * indicates pictures where p65 and ING4 staining were merged; ‡ indicates pictures where p65, ING4, and DAPI staining were merged.