Journal: The Journal of Cell Biology

Article Title: KIF14 controls ciliogenesis via regulation of Aurora A and is important for Hedgehog signaling

doi: 10.1083/jcb.201904107

Figure Lengend Snippet: AURA activity mediates effects of KIF14 depletion on primary cilia formation. (A) Experimental design of KIF14 siRNA effects rescue using 100 nM TCS7010 (AURA inhibitor). (B–J) hTERT RPE-1 cells were transfected with indicated siRNA 48 h before fixation and last 24 h serum starved and AURA inhibited by TCS7010. (B and C) Examination of FBF1 (green) localization and intensity. Representative images (CAP350 in red; scale bar, 1 µm) are shown in B and intensity quantification (normalized to CAP350) in C. (D and E) Examination of SCLT1 (green) localization and intensity. Representative images (CAP350 in red; scale bar, 1 µm) are shown in D and intensity quantification (normalized to CAP350) in E. (F and G) Examination of IFT57 (green) localization and intensity. Representative images (CAP350 in yellow, Ac-tub in red; scale bar, 2 µm) are shown in F and intensity quantification histograms (normalized to CAP350; N = 5) in G ("norm." means normalized to CAP350). (H) Representative images of AURA inhibition rescue experiment of ciliogenesis defect caused by KIF14 knockdown. Detection of ARL13B + primary cilia (green; γ-tubulin, red; DNA, blue); scale bar, 10 µm. (I) Quantification of ARL13B + primary cilia formation. (J) Effects on ARL13B + primary cilia length. Asterisks or "ns" indicates statistical significance determined using Tukey's multiple comparisons test (C, E, and J), an unpaired t test (I; ciliated cells = short + long), or the Holm–Sidak method (I; categories).

Article Snippet: Where indicated, cells were treated for indicated time period with either vehicle (DMSO) or the following compounds at the indicated dose: 100 nM TCS7010 (AURA inhibitor; Tocris Bioscience; ; and ), 10–20 μM (R)-roscovitine ( ; ), or 10 μM monastrol ( ).

Techniques: Activity Assay, Transfection, Inhibition, Knockdown

Journal: The Journal of Cell Biology

Article Title: KIF14 controls ciliogenesis via regulation of Aurora A and is important for Hedgehog signaling

doi: 10.1083/jcb.201904107

Figure Lengend Snippet: KIF14 regulates HH signaling independently of AURA activity. (A–C) Functional analyses of HH pathway activation following control or KIF14 silencing and mock or 2 µM SAG treatment in nHDFs. (A) Quantitative RT-PCR quantification of the effect of GLI1 on mRNA levels . (B and C) Analysis of the effect of KIF14 depletion on SMO translocation to the ciliary axoneme and its intensity. (B) Representative images of SMO (green) and ARL13B (red) IF detection; scale bar, 2 µm. (C) Quantification of changes in relative SMO intensity. (D–F) Examination of effect of TCS7010 AURA inhibition on response of nHDF cells transfected with the indicated siRNA to HH pathway activation. Experimental setup was the same as in , but with an additional 0.5 µm SAG treatment for the last 24 h. (D) Representative images of SMO (green) and ARL13B (red) IF detection in nHDF cells transfected with ctrl or KIF14 siRNA and treated with mock or AURA inhibitor; scale bar = 2 µm. Quantification of changes in SMO intensity (normalized to ARL13B) is shown in E and quantitative RT-PCR quantification of effect on mRNA level of GLI1 in F. Asterisks or “ns” indicate statistical significance determined using Tukey's multiple comparisons test.

Article Snippet: Where indicated, cells were treated for indicated time period with either vehicle (DMSO) or the following compounds at the indicated dose: 100 nM TCS7010 (AURA inhibitor; Tocris Bioscience; ; and ), 10–20 μM (R)-roscovitine ( ; ), or 10 μM monastrol ( ).

Techniques: Activity Assay, Functional Assay, Activation Assay, Control, Quantitative RT-PCR, Translocation Assay, Inhibition, Transfection