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. 2017 Nov 27;7:16373. doi: 10.1038/s41598-017-16693-8

Figure 6.

Figure 6

The enhancement of AMPKα1 activation by minocycline was involved in its effects on radiation-induced autophagy and apoptosis in HT22 cells. (a) Minocycline enhanced AMPKα1 activation after radiation. Cells with and without minocycline pretreatment were collected at different times post IR and lysed. The expression levels of phosphorylated AMPKα1 and AMPKα1 were determined by western blot. p-AMPKα1 and AMPKα1 were probed on the same part of PVDF membrane after stripping, β-actin was probed from different part of the same membrane. (b) A769662, a commonly used AMPK activator, enhanced radiation-induced AMPKα1 activation and LC3 II expression, but decreased cleaved caspase-3 levels. Cells pretreated with minocycline and/or A769662 were collected 48 h after 12 Gy of X-irradiation, and lysed. The expression levels of phosphorylated AMPKα1, AMPKα1, LC3 II and cleaved caspase-3 were determined by western blot. p-AMPKα1 and AMPKα1 were probed on the same part of PVDF membrane after stripping, LC3 and cleaved caspase-3 were probed on the another same part of membrane after stripping, β-actin was probed from different part of the same membrane. (c) A769662 increased radiation-induced autophagy, which was similar to minocycline. Cells pretreated with minocycline and/or A769662 were stained with AO 48 h after irradiation and analyzed on a flow cytometer. (d) A769662 showed the tendency to prevent radiation-induced apoptosis. Cells pretreated with minocycline and/or A769662 were collected 48 h after irradiation and assayed with Annexin V-FITC apoptosis detection kit. (e) AMPKα1 knockdown caused a defect in AMPKα1 activation induced by minocycline and radiation. HT22 cells were infected with virus with A and B shRNAs for 24 h, then treated with minocycline and X-irradiation. Proteins were extracted 48 h post IR, and the expression levels of phosphorylated AMPKα1, AMPKα1, LC3 II and cleaved caspase-3 were determined by western blot. p-AMPKα1 and AMPKα1 were probed on the same part of PVDF membrane after stripping, LC3 and cleaved caspase-3 were probed on the another same part of membrane after stripping, β-actin was probed from different part of the same membrane. (f) AMPKα1 knockdown abolished the enhancive effect of minocycline on radiation-induced autophagy. Cells infected with virus with A and B shRNAs were treated with minocycline and radiation, the percentage of autophagic cells was assessed by AO staining 48 h later. (g) AMPKα1 knockdown significantly mitigated the inhibitory effect of minocycline on radiation-induced apoptosis. Cells infected with virus with A or B shRNAs were treated with minocycline and radiation, apoptosis was then evaluated using Annexin V-FITC apoptosis detection kit 48 h later. *P < 0.05, **P < 0.01 compared with the relative control.