Figure 5: WDR44 displays serum- and Akt-dependent binding to Rab11a, and its knockdown promotes ciliogenesis to the CV stage.

(A) Total peptide count from mass spectrometry analysis of Rab11a-binding proteins immunoprecipitated with GFP antibodies from stable GFP-Rab11a RPE-1 cells grown in the presence or absence of 10% serum for 1h. Normalized peptide count was obtained by multiplying peptide count in - serum lane by a factor of 1.29 obtained after normalizing Rab11a peptides.

(B) Quantification of RPE-1 GFP-Rabin8 centrosomal trafficking in presence of 10% serum following a 72h siRNA treatment and imaged as described in Fig 1F. Trafficking after 1h in absence of serum for siControl is also shown. Mean ± s.e.m from three independent experiments is shown. **P < 0.01, *P < 0.05.

(C) Immunoprecipitation of endogenous WDR44 from RPE-1 GFP-Rab11a cells. Cells were grown in the presence of 2% serum, in serum-starved conditions, or treated with LY294002 (150μM) or AKTiIV (50nM) in 2% serum before immunoprecipitation with WDR44 antibody. Immunoblotting was performed with GFP, WDR44, and pAkt-substrate antibodies. Representative blot from three experiments is shown.

(D) Spinning-disk confocal imaging of transiently-expressed GFP-WDR44 in RPE-1 cells grown with or without serum following PFA fixation and staining with anti-Rab11 and secondary Alexa-568 antibodies. White box in full-cell, “DAPI + merged” images shows magnified area of the cell. Scale bar = 10μm.

(E) (Top) Western blot corresponding to cells treated with control [C] or WDR44 siRNAs [#1 and #2] for 72h in serum and probed with WDR44 and actin antibodies. (Bottom) Quantification of ciliation in cells treated with control or WDR44 siRNAs for 72h in 10% serum and stained as described in Fig 1A. Cells were also treated with control siRNAi for 72h and serum-starved for 24h prior to fixation. Mean ± s.e.m from three independent experiments is shown. n>100 cells counted per treatment. ***P < 0.001, *P < 0.05.

(F) (Left) Representative images showing CP110 loss upon WDR44 knockdown in serum-fed RPE-1 cells as described in (E) and stained and quantified (Right) as described in Fig 1D. Mean ± s.e.m from three independent experiments is shown. *P < 0.05. Scale bar = 2μm.

(G) GFP or non-targetable (NT) GFP-WDR44 plasmids were transfected into RPE-1 cells after 24h of siRNA transfection. 48h later, cells were stained, and CP110 loss was quantified as described in (F). Mean ± s.e.m from three independent experiments is shown. **P < 0.01.

(H) Electron micrographs (Left) of serum-fed RPE-1 cells treated with siControl and siWDR44 for 72h. (Right) Quantification of ciliary vesicle (CV), distal appendage vesicles (DAV), and mother centriole (MC) in EM images. Mean ± s.e.m from three independent experiments is shown for siControl n=62 and siWDR44 n=59 treated cells where the mother centriole distal ends could be observed. For MC, * P <0.05; for CV, ** P < 0.001. Scale bar = 200nm.

(I) Quantification of ciliation in GFP Rab8aQ67L RPE-1 cells treated with WDR44 siRNAs in serum for 72h. Mean ± s.e.m from three independent experiments is shown. **P < 0.01.

See also Figure S5.