Figure 5

Thymosin beta-4 expression was inhibited in Nrf2-knockdown (KD) cells. (A–G) HeLa cells were treated with retroviral shNrf2. Then, shNrf2-positive stable cells were selected by the treatment with hygromycin. Nrf2-KD was induced by the incubation with doxycycline(Dox) for 2 days. Nrf2 proteins were detected by western blotting (A). Wildtype (WT) control and Nrf2-KD HeLa cells were treated with DCF-DA. ROS production was measured by FACS analysis (B, left). Geometric mean fluorescence intensity (MFI) was analyzed by WinMDI 2.8 for each sample (B, right). WT control and Nrf2-KD HeLa cells were transfected with antioxidant response element (ARE)-Gaussia luciferase (Gluc) plasmid DNA and incubated in the absence or presence of 50 μM H₂O₂. ARE-Gluc activity in cultured media was measured with luminometer using Gluc substrate (C). WT control and Nrf2-KD HeLa cells were incubated with 50 μM H₂O₂ for 15 and 30 min. Nrf2 proteins were detected by western blotting (D). The cells were fixed and stained with antibody against Ac-tubulin. The ciliated cells out of more than 1000 cells were counted in control (white) and Nrf2-knockdown (grey) group. PC frequency was evaluated by the blinded double scoring (E). TB4 transcripts were detected with RT-PCR (F). WT control and Nrf2-KD HeLa cells were transfected with pEZX-PG02-TB4-promoter Gaussia luciferase (Gluc) plasmid and incubated in the presence or absence of FBS. The activity of Gluc in cultured media was measured with luminometer using Gluc substrate (G). Each result was the representative of experiments performed at least four times and each experiment was performed with four samples (n = 4) in the same group. Processing (such as changing brightness and contrast) is applied equally to controls across the entire image. Original images of full-length blots were included in the Supplementary Information file (A, D). Data in bar graphs represents the means ± SD. **p < 0.01; significantly different from H₂O₂-untreated and ARE-untransfected or -transfected group (C) or 5% FBS-treated group (G). ^&p < 0.05; ^&&p < 0.01, significantly different from shNrf2-untreated WT control cells (B, right, C, E, G).