Fig. 3: CNPY4 intersects the HH pathway at the level of SMO.
From: CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids
a Immunofluorescence-based staining of primary cilia (acetylated tubulin, red), SMO (SMO, green), and nuclei (DAPI, blue) in ciliated NIH3T3 cells treated with control or Cnpy4 siRNA. The scale bar represents 10 μm. Cilia scale bar represents 1 μm. b Quantification of the percentage of ciliated NIH3T3 cells, as assessed by acetylated tubulin immunofluorescence. Data represent the mean ± SEM (n = 152 siCtrl cells and n = 110 siCnpy4 cells from three biological replicates). Significance was calculated using a two-sided unpaired Welch’s t-test with ns p > 0.05 (p = 0.5286). c Quantification of the length of cilia in NIH3T3 cells. Measurements were made in FIJI using the acetylated tubulin channel. Data represent the mean ± SEM (n = 77 cells from three biological replicates). Significance was calculated using a two-sided unpaired Welch’s t-test with ***p < 0.001 (p = 0.0002). d–g Luciferase reporter assay in ciliated Ptch1 (d), Sufu (e), or Smo (f, g) null MEFs treated with control (gray bars) or Cnpy4 (blue bars) siRNA. Smo null MEFs were stimulated with either SAG (f) or SHH (g). h, i Luciferase reporter assay in ciliated NIH3T3 cells treated with control (gray bars) or Cnpy4 (blue bars) siRNA stimulated with SAG (h) or SHH (i) in the presence of SMO antagonist (SANT-1). Data for d–i were normalized to the average value of control siRNA treated cells stimulated with DMSO where applicable. Data represent the mean ± SD (n = 9 from three biological and three technical replicates). Significance was calculated using a two-sided Mann–Whitney non-parametric test with ns p > 0.05 (psiCtrl+SAG+SANT1 = 0.0503), **p < 0.005 (psiCnpy4+SHH+SANT1 = 0.0040), ****p < 0.0001. Experiments were performed a minimum of three independent times with similar results.
