The effect of GA on NF-κB and on apoptosis are mediated through the transferrin receptor. (A) Down-regulation of TfR1 by RNA interference reverses the effect of GA. A293 cells were transfected with TfR1 si RNA or scrambled (SC) control. After 48 hours, cells were harvested and used for RNA isolation and for protein extraction. RT-PCR was done to determine the TfR1 mRNA expression. RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Whole-cell extracts were analyzed by Western blotting with an anti-TfR1 antibody. (B) Transfected cells were preincubated with 5 μM GA for 4 hours and then treated with 0.1 nM TNF for 30 minutes. The nuclear extracts were prepared and assayed for NF-κB activation by EMSA. (C) Transfected cells were treated with 1 μM GA for 4 hours followed by 1 nM TNF for 24 hours. The cells were stained with a live/dead assay reagent and analyzed under a fluorescence microscope.