Fig. 3

Possible role of LKB1 as an upstream kinase of AMPK in zinc neurotoxicity. a Representative western blots and quantitation of the phosphorylation and expression of LKB1 over the time-course of zinc treatment (n = 3/group, *p < 0.05 compared to sham controls, two-tailed t-test). Protein samples were prepared from zinc-treated (300 μM for 10 min), near-pure neuronal cultures at the indicated time points. b Microscopic images (left) and quantitative data (right, mean ± SEM, n = 3 cultures) of Hoechst 33342-stained total nuclei (upper) and TUNEL-positive apoptotic nuclei (lower) of the same field in mouse cortical near-pure neuronal cultures at 9 h after a 10-min exposure to 300 μM zinc with or without siRNA knockdown with three different LKB1 siRNAs (#1, #2 or #3); scrambled siRNA was the negative control (NC). Arrows indicate apoptotic nuclei. Scale bar, 25 μm. *p < 0.05 compared to zinc-exposed cultures, two-tailed t-test. c Representative western blots and quantitation of the expression levels of LKB1 and phosphorylation of AMPK (n = 3/group, *p < 0.05 compared to zinc-exposed cultures). Near-pure cortical neuronal cultures were transfected with siRNAs at DIV3 for 48 h, and then excess zinc was added (300 μM for 10 min). Protein samples were prepared at 3 h after zinc treatment. d Quantitative data for TUNEL-positive apoptotic cells (mean ± SEM, n = 4 cultures) in mouse cortical near-pure neuronal cultures at 9 h after a 10-min exposure to 300 μM zinc with or without siRNA alone or siRNA plus cDNA of active form of human AMPKα1 or AMPK α2. Introduction of the active form of AMPKα1/α2 reversed neuroprotection by LKB1 siRNA #1 and significantly increased neuronal death in LKB1 siRNA #3 knockdown condition. e Representative western blots and quantitation of phosphorylation and expression levels of LKB1 (n = 3/group, *p < 0.05 compared to zinc-exposed cultures). Protein samples were prepared from cortical neuronal cultures at 0.5 h after 300 μM zinc for 10 min with or without 5 μM GF109264X (GFX), a chemical inhibitor of PKC. The phophorylation and expression levels of LKB1 induced by zinc were reversed by PKC inhibition