Fig. 3.

Septins localize to the axoneme of long mature cilia in RPE1 cells. (A) RPE1 cells maintained in the presence of serum (S) or grown in low serum for 24 (24 h) or 48 (48 h) hours were lysed and expression of SEPT2, SEPT7 and SEPT9 was analyzed by western blotting with the indicated antibodies, expression of γ-tubulin was also analyzed in the same conditions as a control. (B,C) RPE1 cells, serum-starved for 6 (B) or 48 hours (C), were processed for immunofluorescence using antibodies against acetylated tubulin (AcTub; green) and anti-SEPT9 (red). Similar results were obtained using an anti-SEPT7 antibody (supplementary material Fig. S2). Panels on the right are enlarged views of representative cilia (boxed in the main images). (D,E) RPE1 cells were transiently transfected with plasmids encoding SSTR3–GFP (green) and SEPT9–tomato (red) fusions, serum-starved for 48 hours and directly analyzed by epifluorescence microscopy. Representative short (D) and long (E) cilia are shown. Panels on the right are enlarged views of each cilium (white boxes). Arrows and arrowheads indicate the distal end of SEPT9 and SSTR3 stainings, respectively. (F) RPE1 cells, serum-starved for 24 hours were processed for immunofluorescence using a mix of mouse monoclonal antibodies against acetylated-tubulin and γ-tubulin (AcTub+γTub; blue), a goat polyclonal against NPHP1 (green) and a rabbit polyclonal against SEPT7 (red). Panels on the right are enlarged views of a representative cilium (boxed in the main images) where the white arrows indicate NPHP1 staining. (G) RPE1 cells starved for 48 hours were stained for acetylated-tubulin (AcTub, green) and SEPT7 (red). Stacks of images were deconvoluted (left panels) and 3D reconstruction images (right) showed that SEPT7 staining overlaps with AcTub. Scale bars: 5 µm.