Fig. 3

Increased mitochondrial ROS levels mediate primary ciliogenesis by depleting HSPA9 levels in SH-SY5Y cells. (A) SH-SY5Y cells transfected with scrambled siRNA (Sc) or siRNA against HSPA9 (siHSPA9) for 3 days were further treated with N-acetylcysteine (NAC, 1 mM), and then incubated with DCFH-DA. The cells were imaged (left), and the fluorescence intensity of DCF was measured using a fluorescence microplate reader (right). (B) SY5Y/Mito-HyPer cells were transfected with Sc or siHSPA9, and then treated with Mito-Q (20 nM). The level of mitochondrial H₂O₂ was imaged (left; scale bar, 5 μm) and measured using the fluorescence intensity of Mito-HyPer (right). (C, D) SH-SY5Y cells were transfected with Sc or siHSPA9 for 3 days and treated with or without NAC (1 mM) or Mito-Q (1 µM). The cells were then immunostained with ARL13B (green) and Hoechst 33,342 dye (blue). Representative cilia images are presented (C) (Scale bar, 5 μm). Cilia measurement data were obtained from approximately 200 cells per group (D). The experiments were repeated at least three times. Data are presented as the mean ± SEM. (n = 3, **** p < 0.001)