Figure 3

Expression of nucleolin, a Tipα receptor, is reduced by BTG2 expression via the inhibition of Sp1 binding to the nucleolin promoter. To investigate whether BTG2 regulates the expression of the Tipα receptor, nucleolin (NCL) mRNA was isolated from MKN-1 cells infected with either Ad-LacZ or Ad-BTG2 before the following analyses: (a) RT-qPCR, in which BTG2 reduced NCL mRNA expression in MKN-1 cells compared with that in LacZ-transduced cells. GAPDH was used as an internal control. (b) IB analysis, in which BTG2 significantly reduced NCL protein expression. α-Tubulin served as a loading control. (c) RT-qPCR analysis, in which NCL expression was lower in the BTG2 expresser than in the LacZ expresser independent of Tipα treatment. (d) RT-qPCR analysis revealed the inhibition of Sp1 expression in MKN-1 cells with BTG2 expression. GAPDH was internal control. (e) Chromatin immunoprecipitation (ChIP) assay. Sp1 binding to the NCL promoter was reduced in the BTG2-transduced MKN-1 cells compared with that in the LacZ-transduced cells. Immunoprecipitation (IP) with normal IgG was employed as the IP control (Left panel). ImageJ analysis was used to analyze Sp1 binding to the NCL promoter in the LacZ- and BTG2-transduced cells. BTG2 transduction significantly downregulated Sp1 binding to the NCL promoter (right panel). (f) IB analysis revealed the knockdown of BTG2 expression by transfection of MKN-1 cells with siBTG2. siControl RNAs were obtained from the scrambled sequences. (g) ChIP assay confirmed the activity of the BTG2 gene in Sp1 binding to the NCL promoter (upper panel). ImageJ analysis was applied, and we found that Sp1 binding was significantly reduced in the BTG2 expresser but was recovered by transfection with siBTG2 (lower panel).