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. 2016 May 20;24(15):839–854. doi: 10.1089/ars.2014.6128

FIG. 5.

FIG. 5.

The effect of DATS on the expression and function of MT2A, as well as the relationship with NF-κB in GC cells. BGC823 cells were transfected with ectopic MT2A for 36 h, followed by 40 μM DATS treatment for 12 h (A), or BGC823 cells were treated with 40 μM DATS for 6 h, and recultured in fresh complete medium for transfection of shMT2A for 48 h (C). Cell extracts were immunoblotted for determination of MT2A, IκB-α, p65, CCND1, and p-IκB-α. Flow cytometry of apoptosis in BGC823 cells transfected with ectopic MT2A (B) or shMT2A (D). Cells transfected with control vector (Pc) or shMT2A-2 were used as negative controls. Left panel: representative results; Right panel: summary of apoptosis assays, presented as the mean ± standard deviation (n = 3), **p < 0.01 versus Pc control (B) or Ps+DATS (D). (E) A schematic illustration of IκB-α promoter regions constructed into luciferase reporters: pIκB-α-Luc (−2kb/0 from TSS), pIκB-α-4-Luc (−494/−212 from TSS), and pIκB-α-5-Luc (−308/+181 from TSS), and the promoter regions covered by ChIP primers, including the primer set 4 (P4) (−494/−212 from TSS) and primer set 5 (P5) (−308/+181 from TSS). The sequences and the positions of luciferase reporter constructs and ChIP primers are shown in Supplementary Table S1. (F) Luciferase reporter assay showing IκB-α activities in BGC823 cells transfected with IκB-α promoter construct, pIκB-α-Luc, for 36 h, followed by 12 h of treatment with 40 μM DATS or saline. The data are presented as the mean ± standard deviation (n = 3), *p < 0.05 versus control. (G) ChIP assay to identify in vivo association of DATS-induced MT2A with IκB-α promoter using antibody specific for MT2A, or normal IgG, followed by PCR amplification with primers specific for different IκB-α promoter regions (P1 to P5). Chromatin (1% of input) was used as input control. PCR products were visualized on a 1.5% agarose gel. PCR products amplified by ChIP primer sets 4 and 5 are shown. (H) Luciferase assay measuring IκB-α activities in BGC823 cells transiently cotransfected with pIκB-α-4-Luc or pIκB-α-5-Luc. pcDNA3.1-MT2A and pRL-TK serve as internal controls. pGL-3-control vector and pGL-3-basic vector serve as positive and negative controls. The experiments were repeated at least thrice, and the results are presented as the mean ± standard deviation, * p < 0.05, versus control. CCND1, cyclin D1.