Immunofluorescence Analysis of Ciliogenesis

Cells were fixed with cold methanol for 5 min. VPS39-silenced cells were fixed 96 h after the silencing while VPS39-overexpressing cells were fixed 72 h after transfection, blocking and permeabilization were achieved in 0.2% Triton X-100, 10% FBS in PBS. For LC3 detection blocking/permeabilization solution was performed with 0.5% bovine serum albumin (BSA), 0.05% saponin, 50 nM NH4Cl, 0.02% NaN3, in PBS 1X, pH 7.2–7.4). Primary antibodies were: ARL13b (17711-1-AP, Proteintech 1:1000), γ-tubulin (GTU-88, Sigma-Aldrich 1:5000), FLAG-M2 (F3165, Sigma-Aldrich 1:250), IFT20 (13615-1-AP, Proteintech 1:100), OFD1 (HPA031103, Sigma-Aldrich 1:800), LC3B (NB100-2220, Novus Biologicals 1:400). Secondary antibodies Alexa Fluor® IgG were from Thermo Fisher Scientific (1:800). Hoechst 33342 (14 522, Sigma-Aldrich 1:1000) was used to stain nuclei. Confocal fluorescence microscopy and image processing were performed, as described in (72 ). The significance of the results was calculated by Student’s t-test or Anova and reported as P-value.
Definition of the pericentriolar region. We acquired images by confocal microscopy and drew a cube of 2 μm³ around the centrosome to define the pericentriolar region, as described in (73 (link)). The acquisition of z-stack imagines was performed collecting 6 slices for sample.