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

Figure 3.

Figure 3

Minocycline displayed complicated effects on DDR, but did not obviously facilitate DNA damage repair in irradiated HT22 cells. (a) Western blotting showing that minocycline pretreatment inhibited ATM activation in irradiated HT22 cells. PVDF membrane was first probed with p-ATM antibodies, then stripped and probed with ATM antibodies. (b) Western blotting showing that minocycline did not have any effect on p53 activation in irradiated HT22 cells. p53 and GAPDH were probed from different parts of the same PVDF membrane. (c) Western blotting showing that minocycline pretreatment inhibited the induction of γ-H2AX in irradiated HT22 cells. Irradiated HT22 cells with/without minocycline pretreatment were collected at different times and lysed, the proteins of interest were detected by western blot. γ-H2AX and β-actin were probed from different parts of the same PVDF membrane. (d) Minocycline increased radiation-induced G2/M arrest in HT22 cells. **P < 0.01 compared with the relative control. Irradiated HT22 cells in the presence or absence of minocycline pretreatment were collected and fixed 24 h post IR. Cell cycle distribution was measured using flow cytometry. (e) Minocycline did not affect the kinetics of appearance and disappearance of 53BP1 foci in irradiated HT22 cells. Irradiated HT22 cells with and without minocycline pretreatment were fixed at different times after radiation, immunostained with 53BP1 antibodies and observed under fluorescent microscope. Representative fluorescence images of 53BP1 foci in HT22 cells 30 min post IR are shown in left panel. The positive cells were defined as the cells with 5 or more 53BP1 foci. The percentage of positive cells were calculated. Quantification of positive cells with 53BP1 foci is shown in right panel. (f) Left panel: the representative images of comet tail of a single cell from different groups at 30 min post IR; Right panel: distribution of radiation-induced tail moments in HT22 cells with/without minocycline pretreatment at 30 min and 24 h post IR. HT22 cells treated differently were collected at different times and assayed by neutral comet assay.