Figure 6: Actin remodelling factors LIMK2 and TESK1 repress ciliogenesis.

(a) Quantification of ciliogenesis in RPE1 cells transfected with pooled siRNAs targeting kinases involved in actin dynamics for 72 h. Cells were serum-starved for final 48 h. (b) Immunoblotting to confirm siRNA-mediated knockdown of LIMK2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (c) qRT–PCR to confirm siRNA-mediated depletion of TESK1 transcripts. GAPDH was used as a normalization control. (d) Quantification of ciliogenesis in cells transfected with LIMK2 and TESK1 siRNAs for 72 h without serum starvation. (e) Quantification of ciliated cell frequency in cells transfected with flag-tagged TESK1 vector. Cells were serum-starved for final 24 h. (f) Immunofluorescence image displaying the effect of LIMK2 and TESK1 knockdown on ciliogenesis in cells arrested in early S phase using a single thymidine block for 36 h before fixation. Scale bar is 20 μm. (g) Immunoblot analyses of CFL and phospho-CFL after LIMK2 or TESK1 knockdown. GAPDH was used as a loading control. (h) Immunoblotting to confirm siRNA-mediated knockdown of CFL. GAPDH was used as a loading control. (i) Quantification of ciliogenesis in cells depleted with CFL and serum-starved for 24 h. (j) Quantification of ciliated cell percentage in cells transfected with indicated siRNAs without serum starvation. All error bars are s.d. (a,c, n=2; d,e,i,j, n=3 independent experiments). The P-values were determined by unpaired Student’s t-test; *P<0.05, **P<0.01.