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

Figure 5.

Figure 5

The apoptosis-inhibitory effect of minocycline was associated with its enhancive effect on autophagy. Autophagy inhibitor, 3-MA, abolished the protective effect of minocycline on radiation-induced apoptosis in HT22 cells. (a) The representative fluorescence images of AO in HT22 cells with different treatment (left panel) and quantification of autopahgic cells in irradiated cells with and without minocycline and 3-MA (right panel). Cells were stained and analyzed 48 h after irradiation. (b) Immunoblots for LC3 and cleaved caspase-3 in irradiated HT22 cells with and without minocycline and 3-MA. Cells were collected and lysed 48 h post IR, expression levels of LC3 II and cleaved caspase-3 were then determined by western blot. LC3 and cleaved caspase-3 were probed on the same part of PVDF membrane after stripping, β-actin was probed from different part of the same membrane. (c) Quantification of radiation-induced apoptosis in the presence or absence of minocycline and 3-MA. Cells were collected 48 h post IR and assayed with Annexin V-FITC apoptosis detection kit. ATG7 knockdown abolished the protective effect of minocycline on radiation-induced apoptosis in HT22 cells. (d) Immunoblots for Atg7, LC3 and cleaved caspase-3 in HT22 cells infected with lentiviral particles with scramble control shRNA (NC) and ATG7-targeting shRNA constructs (#1 and #2) followed by different treatment. Cells were infected and irradiated with and without minocycline, 48 h after irradiation, cells were collected and lysed, the proteins of interest were detected by western blot. LC3 and cleaved caspase-3 were probed on the same part of PVDF membrane after stripping, Atg7 and β-actin were probed from different parts of the same membrane. (e) Quantification of autopahgic cells in ATG7-knockdown cells 48 h after exposure to X-irradiation in the presence or absence of minocycline. Forty-eight hours after irradiation, ATG7-knockdown cells were stained with AO and analyzed by flow cytometry. (f) Quantification of radiation-induced apoptosis in ATG7-knockdown cells in the presence or absence of minocycline. Cells were collected 48 h post IR and assayed with Annexin V-FITC apoptosis detection kit. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the relative control.