Figure 1.

MRTF and SRF are necessary for serum-induced primary cilium resorption

(A) LLC-PK1 cells were transfected with control (non-related, NR) or specific siRNAs against pig MRTF A/B (siMRTF) or SRF (siSRF) (50 nM for each gene) for 24 hr under serum-free conditions. The cells were then exposed to fresh serum-free or 10% serum-containing medium (serum) for 24 hr. Primary cilia were visualized by acetylated tubulin staining (red) and nuclei by Dapi (blue). Bar: 20 μm.

(B) Effective silencing of MRTF and SRF in LLC-PK1 cells was verified by immunoblotting.

(C) Time course of serum-induced primary cilium resorption in LLC-PK1 cells transfected with NR, siMRTF or siSRF. The percentage of ciliated cells was determined using acetylated tubulin staining for each time point (0, 4, 8, 12, 24 hr); n = 5–6 for each time point, with ≈150 cells counted/condition. The plot shows each individual point, and the mean ± SEM. The percentage of ciliated NR-transfected cells was compared to that of siMRTF- or siSRF-transfected cells at each corresponding time point using unpaired t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

(D) Human retinal pigment epithelial cells were transfected with siNR and human siMRTF and treated and stained as in A.

(E) MRTF knockdown was verified by western blotting.

(F) Time course of ciliation percentage, quantified as in C, n = 6.

(G and I) Verification of the effect of MRTF silencing on serum-induced cilium resorption using an alternative cilium marker, Arlb13 in LLC-PK1 (G) and RPE (H) cells. Conditions were as in A.

(H and J) Quantitation of the results shown in G and I, respectively; n = 4, groups were compared by ANOVA.