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. 2020 Jul 23;21(15):5239. doi: 10.3390/ijms21155239

Figure 4.

Figure 4

Select pro-apoptotic members of the Bcl-2 family are upregulated in irradiated primary cortical neurons. Post-ChIP qPCR to examine occupancy of p53 on Noxa promoter region (A). (T(4) = 2.08, p = 0.0001). Data presented as fold change to non-irradiated control group. n = 3/group. Significance assigned based on one-tailed t-test, *** p < 0.001 versus control Rat primary cortical neurons (RCNs). qPCR quantification of Noxa (B) (F(9,40) = 63.76, p < 0.0001 for 24 h 2Gy and for 6 h and 24 h 8 and 32Gy, compared to control), Puma (C) (F(9,40) = 56.47, p = 0.0051 for 6 h 2Gy, p = 0.0002 for 30 min 32Gy, and p < 0.0001 for 6 and 24 h 8Gy and 32Gy, compared to control), Bim (D) (F(9,40) = 65.12, p = 0.0044 for 6 h 2Gy, p < 0.0001 for all-time points treated with 8 and 32Gy except 30 min 8Gy, compared to control). Data presented as fold change to non-irradiated control group. n = 5 + /group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control. Irradiation induces dose-dependent neuronal cell death and time- and dose-dependent activation of caspase-dependent apoptosis. Irradiation induces dose-dependent neuronal cell death, and time- and dose-dependent activation of caspase-dependent apoptosis in RCNs. Neurons were irradiated with 2, 8 and 32Gy. Twenty-four hours later, LDH release was measured (E) (F(3,36)= 7.43, p = 0.0175 for 2Gy, p < 0.0001 for 8 and 32Gy). Data expressed as a percentage of levels of non-irradiated control. n = 10/group, * p < 0.05, **** p < 0.0001 vs. control. Neurons were collected 30 min and 24 h after 2, 8 and 32Gy IR. Whole-cell lysates were separated by SDS-polyacrylamide gel and immunoblotted with antibodies against cleaved caspase-3, PARP, α-fodrin, PSD95 and β-actin (F). (G) Quantification of levels of cleaved caspase 3 (F(6,14) = 248.2, p < 0.0001 for 24 h at all doses, compared to control), PARP (F(6,14) = 79.08, p < 0.0001 for 24 h at 8 and 32Gy, compared to control), α-fodrin (120kDa) (F(6,14) = 553.4, p < 0.0001 for 24 h at all doses, compared to control) and PSD95 (H) (F(6,14) = 35.55, p = 0.0087 for 30 min 2Gy, p < 0.0001 at all other doses/times, except 30 min 32Gy, compared to control). n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001 vs. control.