Fig. 5

AMPK-mediated induction of Bim, and resultant activation of caspase-3 in zinc neurotoxicity. (a and b) RT-PCR (a) and western blots (b) to measure the expression levels of Bim, Noxa, and Puma over the time-course of zinc treatment. mRNA and protein samples were prepared at the indicated time points from sham-washed or zinc-treated near-pure neuronal cultures (300 μM for 10 min). Graphed data depict the mean ± SEM levels of Bim/Noxa/PUMA normalized to actin at 4 h or 6 h after zinc treatment (n = 3/group, *p < 0.05 compared to sham control). Noticeable increases in protein levels of Bim, Noxa, and PUMA were observed beginning 3–4 h after zinc treatment. c Western blots for caspase-3 activation. Protein samples were prepared at the indicated time points from near-pure neuronal cultures after zinc treatment. Caspase-3 activation by zinc was clearly observed beginning 5 h after zinc treatment. (d and e) Representative western blots and quantitation of the expression of Bim, Noxa, and Puma (d) and activation of caspase-3 (e) (n = 3/group, *p < 0.05 compared to zinc-exposed cultures). Protein samples were prepared from near-pure neuronal cultures at 4 h for BH3-only Bcl-2 family proteins (d) or 5 h for caspase-3 activation (E) after 10-min exposure to 300 μM zinc with or without 20 μM compound C (CC). Among the BH3-only Bcl-2 family, only the expression of Bim (induced by zinc treatment) was markedly attenuated by Compound C. Caspase-3 activation was also reduced by Compound C in zinc-induced neuronal cell death. f Representative western blots and quantitation of the expression of Bim and activation of caspase-3 (n = 3/group, *p < 0.05). Near-pure cortical neuronal cultures were transfected with siRNAs for LKB1 at DIV3 for 48 h, followed by the addition of excess zinc (300 μM for 10 min). Protein samples were prepared at 4 h after zinc treatment. LKB1 siRNAs #1 noticeably attenuated Bim induction and caspase-3 activation by zinc treatment