Fig 2: Nrf2 downregulation inhibits the expression of downstream target genes NQO1 and HO-1 in ARPE-19 cells. (A) ARPE-19 cells were transfected with siNC and siNrf2 vectors using Lipofectamine® 3000. Cell viability was analyzed by an MTT assay. (B and C) Western blotting was applied to detect the expression of Nrf2, HO-1 and NQO1 proteins. (D) Reverse transcription-quantitative polymerase chain reaction was performed to detect the expression of HO-1 and NQO1 mRNA. *P<0.05, **P<0.01. n=4. HO-1, heme oxygenase-1; NQO1, NAD(P)H: Quinine oxidoreductase; Nrf2, nuclear factor-erythroid 2-related factor-2; siRNA, small interfering RNA; siNC, siRNA negative control; siNrf2, siRNA Nrf2.

Fig 3: Nrf2 silencing enhances H2O2-induced damage to ARPE-19 cells. (A) ARPE-19 cells were subjected to treatment with 200 µM H2O2, NC vector and siNrf2 vector. Flow cytometry was performed to analyze the levels of ROS and the number of apoptotic cells. *P<0.05 vs. H2O2 + NC group. (B) Proteins expression levels of Nrf2, were determined using western blotting. (C) The protein expression levels of NQO1 and HO-1 were detected by reverse transcription-quantitative polymerase chain reaction. (D) The protein expression levels of cleaved-caspase 3, Bax and Bcl-2 were detected by western blotting. (E) The mRNA expression levels of HO-1 and NQO1 were detected by reverse transcription-quantitative polymerase chain reaction. (F) The mRNA expression levels of Bax and Bcl-2 were detected by reverse transcription-quantitative polymerase chain reaction. *P<0.05, **P<0.01. n=4. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X; FITC, fluorescein isothiocyanate; HO-1, heme oxygenase-1; NQO1, NAD(P)H: Quinine oxidoreductase; Nrf2, nuclear factor-erythroid 2-related factor-2; PI, propidium iodide; siRNA, small interfering RNA; NC, siRNA negative control; siNrf2, siRNA Nrf2.

Fig 4: GP suppresses the effects of H2O2 on the levels of ROS and apoptosis of ARPE-19 cells by activating Nrf2 signaling. (A) ARPE-19 cells were exposed to 200 µM H2O2 and 30 µM GP. The ROS levels and apoptosis were assessed by flow cytometry. (B and C) Protein expression levels of Nrf2, HO-1 and NQO1 were evaluated by western blotting. (D) mRNA expression levels of HO-1 and NQO1 were detected by reverse transcription-quantitative polymerase chain reaction. *P<0.05, **P<0.01. n=4. FITC, fluorescein isothiocyanate; GP, genipin; HO-1, heme oxygenase-1; NQO1, NAD(P)H: Quinine oxidoreductase; Nrf2, nuclear factor-erythroid 2-related factor-2; PI, propidium iodide; ROS, reactive oxygen species.

Fig 5: Nrf2 silencing attenuates the effects of GP on H2O2-induced ARPE-19 cells. (A) ARPE-19 cells were subjected to treatment with 200 µM H2O2, 30 µM GP, NC vector and siNrf2 vector. Flow cytometry was applied to analyze the levels of ROS and the number of apoptotic cells. *P<0.05, **P<0.01. (B-D) Proteins expression levels of Nrf2, HO-1, NQO1, Bax, Bcl-2 and cleaved-caspase 3 were determined by western blotting. (E and F) mRNA expression levels of HO-1, NQO1, Bax and Bcl-2 were detected by reverse transcription-quantitative polymerase chain reaction. *P<0.05, **P<0.01. n=4. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X; FITC, fluorescein isothiocyanate; HO-1, heme oxygenase-1; NQO1, NAD(P)H: Quinine oxidoreductase; Nrf2, nuclear factor-erythroid 2-related factor-2; PI, propidium iodide; siNrf2, small interfering RNA Nrf2.