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. 2020 Apr 29;219(6):e201904107. doi: 10.1083/jcb.201904107

Figure S2.

Figure S2.

KIF14 knockdown affects localization of BB components and IFT-B machinery. (A) Validation of KIF14 siRNA knockdown efficiency by Western blot protein expression analysis in Flp-in T-REx RPE-1 cell line (referring to time-lapse imaging experiments in Fig. 3) upon doxycyclin (DOX) induction expression GFP-ARL13B. (B) Quantification of ciliogenesis ability in GFP-ARL13B inducible RPE-1 cells after KIF14 siRNA knockdown. hTERT RPE-1 cells were transfected with the indicated siRNA, serum starved for 24 h, and analyzed by IF microscopy (related to Fig. 4, C–Q). γ-tubulin or CAP350 staining were used to visualize centrosomes, Ac-tubulin or ARL13B staining detected primary cilia. (C–F) Examination of protein levels of BB and ciliary markers used for normalization; levels are not affected by KIF14 siRNA. Relative CAP350 (C) and γ-tubulin (D) BB intensities and relative ARL13B (E) and Ac-tubulin (F) ciliary intensity are quantified in arbitrary units (AU) after background subtraction. (G) KIF14 depletion does not affect process of CP110 removal from the distal end of mother centriole as quantified by number of CP110 dots. TTBK2 knockdown was used as positive control. (H) Representative images of IF staining detects CEP164 (green), γ-tubulin (red), and DNA (blue); scale bar, 2 µm. Quantification of CEP164 intensity on BB (normalized to γ-tubulin) in I (CEP164 siRNA was used as positive control). (J) Representative images of IF staining of IFT88 (green), CAP350 (yellow), and Ac-tub (red); scale bar, 3 µm. (K and L) Quantification of IFT88 intensity (normalized to CAP350) at BBs (K) and a decrease of IFT88 in the ciliary tip (L) are shown. (M) Representative images of IF staining of IFT140 (green), CAP350 (yellow), and Ac-tub (red); scale bar, 3 µm. (N and O) IFT140 intensity (normalized to CAP350) at BBs (N) or in the ciliary tip (O) was not affected by KIF14 depletion. (P–Q) Histograms demonstrate distribution of CAP350 (red), Ac-tub (gray), and IFT (green) proteins along the ciliary axoneme (N = 20). Quantification was done for typical control long cilia and short defective cilia after KIF14 siRNA and for additional control also within a subset of short primary cilia in controls. Asterisks or ns indicates statistical significance determined by an unpaired t test.