Figure 4

Effect of H₂O₂ and Nrf2 on primary cilia formation. (A) Cells were treated with 50 μM H₂O₂ for 15, 30 and 60 min. Cell lysates were prepared from each sample and Nrf2 proteins were detected by western blotting. (B) 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) Cells were treated with 50 μM H₂O₂ in the absence or presence of clobestasol propionate (P.). Cell lysates were prepared from each sample and Nrf2 proteins were detected by western blotting. (D–F) Cells were incubated in the absence or presence of clobestasol P under SD condition. Cells were treated with DCF-DA and ROS production was measured by FACS analysis (D). Geometric mean fluorescence intensity (MFI) in the absence (white) or presence (grey) of clobestasol P. was analyzed by WinMDI 2.8 for each sample (E). The cells were fixed and stained with antibody against Ac-tubulin. The number of ciliated HeLa cells out of more than 1000 cells in the absence (white) or presence (grey) of clobestasol P. were counted. PC frequency was evaluated by the blinded double scoring (F). (G) HeLa cells were transfected with pEZX-PG02-TB4-promoter Gaussia luciferase (Gluc) plasmid and incubated with SD condition in the absence (white) or presence (grey) of clobestasol P. for up to 24 h. The activity of Gluc in cultured media was measured with luminometer using Gluc substrate. 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, C). Data in bar graphs represents the means ± SD (B, E–G). **p < 0.01; significantly different from H₂O₂-untreated and ARE-untransfected group (B) or SD-treated and clobestasol P.-untreated group (E, F). ^&p < 0.05, ^&&p < 0.01; significantly different from H₂O₂-untreated and ARE-transfected group (B) or clobestasol P.-untreated group (G).