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. 2017 Jan 4;8(7):11206–11218. doi: 10.18632/oncotarget.14493

Figure 6. Regulation of autophagy proteins in response to combination of Rad001 and ATO.

Figure 6

For LNCaP cells, they were treated DMSO (control), Rad001 (0.37 μM), ATO (11.19 μM), and their combination, for 24 hrs. For PC3 cells, they were treated DMSO (control), Rad001 (0.35 μM), ATO (12.00 μM), and their combination, for 24 hrs. (A) Atg5-12 conjugates and Beclin1 were induced in response to the treatment of two compounds. (B) Beclin 1 mRNA was more significantly up-regulated by the combination treatment through quantitative RT-PCR analyses in prostate cancer LNCaP and PC3 cell lines. Beclin1 mRNA stability significantly increased in response to the combination treatment of the two compounds. RNA was extracted at the indicated time, and quantitative RT-PCR was used to measure the relative Beclin1 mRNA level compared to that of GAPDH. (C) Beclin1 is critical for the autophagic death induced by the combination of two compounds. LNCaP and PC3 cells were transfected with control siRNA or that for Beclin1. 24 hrs later cells were treated with combination of Rad001 and ATO, and cell survival was gauged with ATP measurement after 24 hrs of combination treatment.