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. 2020 May 19;9(5):1253. doi: 10.3390/cells9051253

Figure 5.

Figure 5

Knockdown of Akt1 potentiates radiation-induced apoptosis in MDA-MB-231 cells. (A) MDA-MB-231 cells were exposed to 0, 6, or 10 Gy radiation dose and collected 48 h or 72 h after irradiation. Expression of cleaved PARP and cleaved caspase-3 was determined by Western blot. (B) MDA-MB-231 cells were exposed to 0, 2, 4, or 6 Gy radiation dose daily for 4 d and collected 24 h after the last radiation treatment. Expression of cleaved PARP and cleaved caspase-3 was determined by Western blot. (C) MDA-MB-231 cells were transfected with siRNA to AMPKα1, Akt1, or AMPKα1/Akt1. Transfection concentrations were (1) individual siRNA: 50 nM, (2) combination siRNA: 50 nM each (100 nM total), and (3) NTC: 100 nM. Cells were exposed to 0 or 6 Gy radiation dose 24 h after transfection and collected 48 h after radiation. Expression of AMPKα1, Akt1, and cleaved PARP was determined by Western blot. (D) MDA-MB-231 cells were exposed to 0 or 4 Gy radiation dose and then transfected with siRNA to AMPKα1, Akt1, or AMPKα1/Akt1 on the same day. Transfection concentrations were (1) individual siRNA: 50 nM, (2) combination siRNA: 50 nM each (100 nM total), and (3) NTC: 100 nM. Cells were collected 72 h after transfection. Expression of AMPKα1, Akt1, and cleaved PARP was determined by Western blot. (E) MDA-MB-231 cells were exposed to 0 or 4 Gy radiation dose and then incubated for 48 h. Cells were then treated with 0 or 10 μM of A-674563 or MK-2206 for 24h. Expression of pAkt, Akt1, and cleaved PARP was determined by Western blot. (F) MDA-MB-231 cells were exposed to 0 or 4 Gy radiation dose and then incubated for 48 h. Cells were then treated with 0 or 10 μM of AZD5363 or perifosine for 24 h. Expression of pAkt, Akt1, and cleaved PARP was determined by Western blot. (G) Summary diagram showing Akt1 inhibition and radiation-induced apoptosis in TNBC cells. For A–D, NTC (non-targeting control) was used as a negative control.