Figure 6. Activation of Akt-mTORC1 is required for D3T-induced Nrf2-HO-1 activation and RPE cytoprotection.

APRE-19 cells were treated with indicated concentrations of D3T, and cultured for indicated time, p-Akt (Ser 473), Akt, p-S6 (Ser 235/236), S6, p-4E-BP1 (Ser 65), 4E-BP1 and tubulin were tested by Western blots, kinase phosphorylations were quantified (A,B). APRE-19 cells were pre-treated with LY294002 (500 nM) or rapamycin (100 nM) for 1 h, followed by UV (30 mJ/cm²), or with D3T (50 μM, 30 min pretreatment), cell were further cultured, Western blots were utilized to test indicated proteins (C), while cell viability and cell death were tested 24 h after UV treatment (D,E). Stable APRE-19 cells expressing scramble-shRNA or Akt1 shRNA were radiated with UV (30 mJ/cm²), and/or D3T (50 μM, 30 min pretreatment), cells were further cultured, expressions of listed proteins were tested by Western blots, and quantified (F), HO-1 mRNA expression was tested (G), cell survival and cell death were also tested by MTT assay (H) and Trypan blue staining assay (I). Experiments were repeated three times, and similar results were obtained. *p < 0.05.