Fig. 1

Depletion of HSPA9 promotes primary ciliogenesis. (A, B) htRPE/Smo-GFP cells were transfected with scrambled siRNA (Sc), or HSPA9 siRNA (siHSPA9). After 3 days, the cells were imaged for Smo-GFP via fluorescence microscopy. Decreased expression of HSPA9 by RNA interference was confirmed via western blotting (A). Scale bar: 10 μm. The ciliated cells and cilium length of the cells were measured (B). (C) RPE cells were transiently transfected with Sc or siHSPA9, and primary cilia were stained with antibodies against ARL13B (green), acetylated α-tubulin (Ac-Tub, red). The nucleus (blue) was counterstained with Hoechst 33,342 dye. And the cells were analyzed by western blotting with indicated antibodies. Scale bar: 5 μm. (D-F) TT cells stably expressing pTRIPZ/shHSPA9 (TT/shHSPA9) were treated with doxycycline (Dox) for 8 days. The cells were stained with ARL13B (green) and Hoechst 33,342 (blue) to count ciliated cells and cilium length (D, E). Scale bar: 5 μm. TT cells treated with Dox were further analyzed by western blotting with anti-IFT88 and anti-acetylated α-tubulin antibodies (F). Data were obtained from at least three independent experiments and the results are presented as the means ± S.E.M. (n = 3, ** p < 0.01, *** p < 0.005, **** p < 0.001)