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. 2021 Feb 26;9:623753. doi: 10.3389/fcell.2021.623753

FIGURE 1.

FIGURE 1

WNT3a does not promote ciliogenesis or cilia length. (A) Experimental scheme of WNT3a treatment experiment. Cells were seeded and grown for 24 h, then starved for additional 48 h. A 2 h treatment (RPE-1) by WNT3a is indicated in blue, 24 h treatment is indicated in orange (RPE-1 and NIH3T3). (B) Western blot analysis of 2 h WNT3a treatment of RPE-1. The treatment leads to LRP6 shift and increased LRP5/6 phosphorylation, DVL2 phosphorylation and upshift, and accumulation of ABC. The quantitation of pLRP5/6 intensity is shown in (C) n = 3, DVL2 band intensities (upper to lower band intensity ratio, the bands are indicated by arrows) is shown in (D) n = 3, the quantification of relative ABC levels is presented in (E) n = 3. (F) Representative images of RPE-1 cells treated by WNT3a or vehicle (control) for 2 h and stained for Arl13b (green) and γ-tubulin (red). Scale bar = 2 μm. DAPI (blue) was used to counter stain nuclei. The corresponding quantification of the cilia length (G) and the percentage of cells with Arl13+ cilium (H). Each dot indicates either length of a single primary cilium (G) or percentage of ciliated cells in a single image (H). (I) Western blot analysis of 24 h WNT3a treatment of RPE-1. The treatment leads to LRP6 shift, increased LRP5/6 phosphorylation, DVL2 phosphorylation and upshift, and accumulation of ABC. The quantification of pLRP5/6 intensity is shown in (J) n = 3, DVL2 bands (indicated by arrows) intensity ratio is shown in (K) n = 3, quantification of relative ABC levels is presented in (L) n = 3. (M) Representative images of RPE-1 cells treated by WNT3a or vehicle (control) for 24 h and stained for Arl13b (green) and γ-tubulin (red). Scale bar = 2 μm. DAPI (blue) was used to counter stain nuclei. The corresponding quantification of the cilia length and the percentage of cells with Arl13+ cilium is shown in (N,O), respectively. Each dot indicates either length of a single primary cilium (N) or percentage of ciliated cells in a single image (O) n = 4. (P) Western blot analysis of NIH3T3 cells treated by WNT3a for 24 h shows LRP6 shift and LRP5/6 phosphorylation, DVL2 phosphorylation and upshift, and accumulation of ABC. The quantification of pLRP5/6 intensity is shown in (Q) n = 3, DVL2 band intensities (upper to lower band intensity ratio, the bands are indicated by arrows) is shown in (R) n = 3, quantification of relative ABC intensity (S) n = 3. (T) Representative images of NIH3T3 cells treated by WNT3a for 24 h, stained for Arl13b (green), and γ-tubulin (red). Scale bar = 2 μm. DAPI (blue) was used to counter stain nuclei. The corresponding quantification of the cilia length (U) and the percentage of cells with Arl13+ cilium (V). Each dot indicates either length of a single primary cilium (U) or percentage of ciliated cells in one image frame (V) n = 3.