Figure 2

N-acetylcysteine (NAC) decreased reactive oxygen species production, primary cilia formation and the expression of thymosin beta-4 under serum-deprived (SD) condition. (A) Cells were treated with 100 μM H₂O₂ in the absence or presence of NAC. Cells were incubated with DCF-DA and ROS production was observed under the fluorescence microscope. (B–D) Cells were incubated under SD condition in the absence or presence of NAC. Cells were treated with DCF-DA and ROS production was measured by FACS analysis. Vertical dotted lines and solid lines indicate mean fluorescence intensity (MFI) for the increase and the inhibition of ROS production, respectively (B). Cells were fixed and stained with antibody against Ac-tubulin (green) and DAPI (blue). The representative image of primary cilia was observed with 400X magnification under fluorescence microscope. White arrows indicated primary cilia (C). The ciliated HeLa cells out of more than 1000 cells in the absence (white) or presence (grey) of NAC were counted. PC frequency was evaluated by the blinded double scoring (D). (E) HeLa cells were transfected with pEZX-PG02-TB4-promoter Gaussia luciferase (Gluc) plasmid and incubated with 5% or 0% FBS for up to 12 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. Data in bar graphs represents the means ± SD. **p < 0.01; significantly different from 5%-treated control group (E). ^&&p < 0.01; significantly different from NAC-untreated group in 0% FBS (D, E).