Figure 5. Nrf2-HO-1 mediates D3T-induced RPE cytoprotective effect against UV.

Stable APRE-19 cells expressing scramble-shRNA (“Ctrl shRNA”) or Nrf2 shRNA (clone-1/-2) were radiated with UV (30 mJ/cm²), or together with D3T (50 μM, 30 min pretreatment), cells were further cultured for 24 h, Nrf2 and tubulin expressions were tested by Western blots, Nrf2 expression was quantified (A), cell viability and cell death were tested by MTT assay (B) and trypan blue staining (D), respectively. The effects of ZnPP (10 μM, 1 h pretreatment) on D3T (50 μM, 30 min pretreatment)-mediated protective effect against UV (30 mJ/cm², cultured for 24 h) in APRE-19 cells were tested by MTT assay (C) and trypan blue staining (D). Stable ARPE-19 cells expressing dominant negative Nrf2 (S40T, DN-Nrf2), wild-type (wt-) Nrf2 or vector (pSV2 puro Flag), as well as the parental ARPE-19 cells were treated with D3T (50 μM) for indicated time, Nrf2 and tubulin expressions were tested by Western blots (E), Nrf2 phosphorylation (vs. Tubulin) was quantified (E); HO-1 mRNA expression was tested (F). Above ARPE-19 cells were radiated with UV (30 mJ/cm²), or together with D3T (50 μM, 30 min pretreatment), cells were further cultured for 24 h, cell survival and cell death were tested (G,H). Experiments were repeated three times, and similar results were obtained. *p < 0.05.