Figure 6
Thymosin beta-4 expression was enhanced by Nrf2-overexpressed cells. (A–D) Cells were transfected with pcDNA3.1 or pcDNA-Nrf2 plasmid DNA for 24 h. Nrf2 proteins were detected by western blotting. 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). HeLa cells were treated with 50 μM of H2O2 for 24. Cells were fixed and stained with antibody against Ac-tubulin (green), Arl13b (red) and DAPI (blue). The representative image of primary cilia was observed with 1000X magnification under fluorescence microscope. White arrows indicated primary cilia (B). The number of ciliated HeLa cells out of more than 1000 cells were counted. PC frequency was evaluated by the blinded double scoring (C). The mRNA expression of TB4 was detected by RT-PCR (D). (E) pEZX-PG02-TB4-promoter Gaussia luciferase (Gluc) plasmids were co-transfected with pcDNA3.1 or pcDNA-Nrf2 plasmid DNA into HeLa cells. Then, cells were incubated in the presence or absence of FBS. 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. Data in bar graphs represents the means ± SD. *p < 0.05, **p < 0.01; significantly different from H2O2-untreated (C) or 5% FBS-treated group (E). &p < 0.05; &&p < 0.01; significantly different from pcDNA3.1-transfected and H2O2-untreated or -treated group (C) or 5% FBS- or 0% FBS-treated group (E). (F) Schematic summary for primary cilia formation by Nrf2 and ROS through TB4 expression to regulate CC survival and death. While Nrf2 conversely regulated primary cilium formation and TB4 expression in HeLa human cervical CCs (solid line). ROS also up-regulate TB4 expression through unknown another factors (marked as question mark and grey dotted line). Our findings were indicated by black solid lines. Pathway already known was indicated by grey solid lines.
