Figure 4

Tipα-induced TNFα expression is inversely regulated by nucleolin and BTG2. To confirm the effect of BTG2 expression on Tipα-induced TNFα expression, endogenous NCL expression was excluded by transfection of MKN-1 cells with siNCL, and then adenoviral transduction was performed. RT-qPCR analysis (a) Tipα-induced TNFα expression was significantly reduced in the BTG2 expresser, but it was recovered by the knockdown of BTG2 with siBTG2 transfection. Expression of GAPDH served as the internal control. (c) IB analysis clearly revealed the regulation of TNFα expression depending on BTG2 expression. Knockdown of nucleolin and BTG2-HA was also revealed. α-tubulin served as an internal control. To explore the effect of NCL on the regulation of Tipα-induced TNFα expression, MKN-1 cells transfected with either siNCL or siBTG2 were transduced with either Ad-LacZ or Ad-BTG2, and then Tipα was treated 1 h before RNA extraction. (b) Note the significant inhibition of Tipα-induced TNFα expression by the knockdown of NCL expression in both the LacZ and BTG2 expressers, indicating the induction of TNFα expression by NCL expression. (d) Immunoblot analysis was performed to analyze the protein expressions of TNFα, nucleolin and BTG2-HA in the present experiment. α-Tubulin served as an internal control. The data strongly suggest that BTG2 and NCL inversely regulate Tipα activity in gastric cancer cells.