Fig. 8.

Effect of sestrin 2 downregulation on the expression of mammalian target of rapamycin (mTOR) in conditionally immortalized cultured PECs. Conditionally immortalized cultured PECs were transfected with short hairpin (sh)RNA targeting sestrin 2 or nontarget control (NTC). Transfected PECs were then cultured under growth-restrictive conditions for 10 days, and proteins were extracted for Western blot analysis. A: Western blot analysis for sestrin 2 and the downstream targets of mTOR. Sestrin 2 expression was reduced in PECs transfected with shRNA targeting sestrin 2 compared with PECs transfected with NTC. Phosphorylation of downstream targets of mTOR [P-4E-binding protein 1 (P-4E-BP1), P-p70 ribosomal protein S6 kinase (P-p70S6K), and P-S6RP] was also increased in PECs transfected with shRNA targeting sestrin 2 compared with PECs transfected with NTC shRNA. B: relative expression of sestrin 2 protein detected by Western blot analysis. Compared with PECs transfected with NTC, a significant reduction of sestrin 2 protein expression was observed in PECs transfected with sestrin 2 shRNA. *P < 0.01. C: relative expression of the proteins downstream of P-mTOR. Expressions of P-4E-BP-1, P-p70S6K, and P-S6RP were significantly increased in PECs transfected with sestrin 2 shRNA compared with PECs transfected with NTC. **P < 0.05. D: Western blot analysis for the downstream targets of mTOR in PECs transfected with shRNA targeting sestrin 2 treated with the AMPK activator aminoimidazole carboxamide ribonucleotide (AICAR). Phosphorylation of the downstream targets of mTOR (P-4E-BP1, P-p70S6K, and P-S6RP) was reduced in sestrin 2 knocked down PECs. F: relative expression of the proteins downstream of P-mTOR. Expressions of P-4E-BP-1, P-p70S6K, and P-S6RP were significantly reduced in PECs transfected with sestrin 2 shRNA with AICAR treatment. *P < 0.01.