Fig. 5: p21 inhibition induces autophagy by triggering intracellular ROS through activation of Akt kinase. | Cell Death & Disease

Fig. 5: p21 inhibition induces autophagy by triggering intracellular ROS through activation of Akt kinase.

From: Inhibition of p21 activates Akt kinase to trigger ROS-induced autophagy and impacts on tumor growth rate

Fig. 5

A Lysates from HCT116 p21wt and null cells were resolved and immunoblotted with indicated Akt-mTOR signaling antibodies. B HCT116 p21+/+ cells were transiently transduced with lentiviral particles encoding control and p21-specific shRNA. Cell lysates were analyzed by western blotting using same sets antibodies as mentioned in panel A. C HCT116 p21wt and null cells were incubated with or without 250 µM tBHP for 24 h in the presence or absence of NAC (2.5 mM NAC; 4 h pre-incubation). Cells were then stained with 5 µM CM-H2DCFDA at 37 °C for 30 min in dark and analyzed by flow cytometry. Data presented as mean of three (n = 3) independent experiments ± SE. D HCT116 p21wt and null cells were incubated with or without 2.5 mM NAC for 4 h followed by immunoblot detection after probing with indicated antibodies. Relative LC3-II expression level among experimental groups was quantified from cell lysates of three independent experiments and graphically represented as mean ± SE. E HCT116 p21+/+ cells were subjected to p21 suppression by sh-p21 lentivirus transduction and co-incubated (for 4 h) with or without 2.5 mM NAC. The cells were then lysed and immunoblotting was carried out to determine relative conversion of LC3 as well as to confirm the silencing of p21 by gene-specific shRNA. Relative LC3-II expression level among experimental groups was quantified from cell lysates of three independent experiments and graphically represented as mean ± SE. F Representative immunoblots of HCT116 p21wt and null cell lysates showing increased phosphorylation of Akt substrates and reduced expression of ROS scavengers in p21 deficient cells. G HCT116 p21null cells were transiently transfected with plasmids encoding or not constitutively activated (myr) Akt for 48 h. Cell lysates were then immunoblotted with indicated antibodies to determine Akt activation and its effect on cellular autophagy. H Quantification of LC3-II turnover by ImageJ in pCDNA and Myr-Akt transfected cells of three independent experiments (mean ± SE; ***P < 0.001). I HCT116 p21null cells, with or without transient expression of myr-Akt, were analyzed by flow cytometry after 30 min staining with 5 µM CM-H2DCFDA.

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