Fig. 2: Absence of Cnpy4 leads to hyperactivation of HH-related gene expression and signaling.
From: CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids
a Shh in situ hybridization and lacZ expression of Cnpy4;Gli1lacZ in hindlimb buds at embryonic day (E)10.5, E11.5, and E12.5. Samples at E11.5 show an enlarged Shh domain (lines) and samples at E12.5 have ectopic expression of both Shh and Gli1 (arrowheads) in Cnpy4 mutants. The scale bars represent 500 μm. b, c Luciferase reporter assay in ciliated NIH3T3 cells treated with control (gray bars) or Cnpy4 (blue bars) siRNA and stimulated with SMO agonist (SAG), recombinant SHH (b), 20(S)-hydroxycholesterol (20(S)-HC), or 24(S), 25-exposycholesterol (24(S), 25-EC) (c). Data were normalized to the average value of control siRNA-treated cells stimulated with DMSO or vehicle. 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 ***p < 0.001 (psiCnpy4+SHH = 0.0004), ****p < 0.0001. d, e qRT-PCR assessment of Gli1 expression in ciliated NIH3T3 cells treated with control (gray bars) or Cnpy4 (blue bars) siRNA and stimulated with SAG, recombinant SHH (d), 20(S)-HC or 24(S), 25-EC (e). Data represent the mean ± SD (n = 12 from three biological and four technical replicates). Significance was calculated using a two-sided Mann–Whitney non-parametric test with ****p < 0.0001. All experiments were performed a minimum of three independent times with similar results.
