Figure S1.

KIF14 knockdown causes ciliogenesis defects in different cell types. (A) Quantitative RT-PCR mRNA expression analysis for validation of siRNA knockdown efficiency (related to Fig. 1 B). For SCYL2 silencing, we used a single siRNA oligo; for LUZP1, CCDC92, KIF7, and KIF14, we used a 50 nM mix of oligos 1 and 2 (Table S3). (B and C) Test of KIF14 knockdown efficiency using different siRNA oligos in hTERT RPE-1 cells. (B) Quantification of Fig. 1 E. Western blot analysis of protein expression in total cell lysates using two different siRNA KIF14 oligos. (C) Quantitative RT-PCR expression analysis of KIF14 mRNA. (D–F) Test of the effect of KIF14 siRNA oligo #3 (Carleton et al., 2006; Table S3) on hTERT RPE-1 cells. (D) Representative image of IF staining of ARL13B (green), CAP350 (red), and DNA (blue); scale bar, 10 µm. (E and F) Graphs of primary cilia formation ability after KIF14 siRNA #3 knockdown (E) and the effect on ARL13B⁺ cilia length (F). (G–I) Validation of the effect of KIF14 knockdown on ciliogenesis in hTERT RPE-1 cells using Ac-tub as a ciliary marker. (G) Representative images of IF staining of Ac-tub (green), CETN1 (red),and DNA (blue); scale bar, 10 µm. Quantification of the experiment is shown as a percentage of ciliated cells (H) and by Ac-tub⁺ cilia length (I). (J) Validation of Flp-in T-REx RPE-1 cell line by Western blot analysis upon DOX induction expressing GFP-KIF14 mutant resistant to KIF14 siRNA 2 (GFP-KIF14siRNA2res; related to Fig. 1, H and I). (K–V) Examination of phenotypes after KIF14 depletion in nHDFs (K–M), hESCs (N–P), hiPSCs (Q–S), and IMCD3s (T–V). (K, N, Q, and T) Representative images of experiments detecting IF staining of ARL13B (green), γ-tubulin (red), DNA (blue); scale bars, 10 µm. Quantifications of experiments (by percentage of ciliated cells) are shown in L, O, R, and U, and graphs showing ARL13⁺ ciliary lengths are shown in M, P, S, and V. Asterisks indicate statistical significance using an unpaired t test.