Induction of caspase 3- and TG2-dependent apoptosis by ACR via a reduction in the expression of EGFR due to silencing of Sp1. A, JHH-7 cells were treated with 10 μM ACR or vehicle for 12 h. Cells were harvested and mRNA expression of indicated genes was determined by RT-PCR. Bar graphs show densitometrically determined relative mRNA abundance normalized to GAPDH mRNA levels. **P < 0.01 compared to each control. B, JHH-7 cells were treated with 10 μM ACR or vehicle for 24 h. Cells were harvested and protein expression of indicated proteins was determined by Western blotting. The bar graph shows densitometrically determined relative protein abundance normalized to GAPDH protein levels. **P < 0.01 compared to each control. C, JHH-7 cells were treated with 10 μM ACR for 24 h in the presence or absence of 100 μM zDEVD, 50 ng/ml EGF or a combination of the two, and the numbers of viable cells were determined after trypsinization by Trypan Blue exclusion. Results shown are means ± SD (n = 3). *P < 0.05, compared to ACR-treated sample from control cells (lane 2). D, One day after transfection of JHH-7 cells with EGFR promoter GC3-Luc (1 μg/dish), cells were treated with 10 μM ACR for 24 h, co-transfected with Sp1 (lanes 3 and 4), TG2 shRNA (lanes 5 and 6), TG2 (lanes 7 and 8), Sp1 shRNA (lanes 9 and 10), and non-target siRNA (lanes 11 and 12) expression vector, and cell lysates were prepared. Luciferase activity of each cell lysate was determined. Results shown are means ± SD (n = 3). *P < 0.05, **P < 0.01 compared to ACR-treated control sample from control cells (lane 2). Panels A-D show representative results from three different experiments with similar results.