Skip to main content
. 2019 Oct 30;8:e50051. doi: 10.7554/eLife.50051

Figure 2. Enzymes that generate cholesterol positively regulate hedgehog signaling.

(A) The post-squalene portion of the cholesterol biosynthetic pathway, with enzymes colored according to their FDR corrected p-value in our CRISPR screens (see Supplementary files 2 and 3). Two branches of the pathway (the Kandutsch-Russell and the Bloch pathways) produce cholesterol, while a third shunt pathway produces 24(S), 25-epoxycholesterol. DHCR24 is the only enzyme that is required for cholesterol biosynthesis, but dispensable for 24(S), 25-epoxycholesterol synthesis. (B and C) HH signaling strength in Lss-/-, Dhcr7-/- and Dhcr24-/- NIH/3T3 cells was assessed by measuring Gli1 mRNA by quantitative reverse transcription PCR (qRT-PCR) after treatment with either HiSHH (25 nM) or HiSHH combined with 0.3 mM cholesterol:MβCD complexes. Bars denote the mean value derived from the four individual measurements shown. Statistical significance was determined by the Mann-Whitney test (B, p-value for HiSHH treatment = 0.0286, p-value for HiSHH + cholesterol treatment = 0.4857) or the Kruskal-Wallis test (C, p-value for HiSHH treatment = 0.0002, p-value for HiSHH + cholesterol treatment = 0.0002).

Figure 2.

Figure 2—figure supplement 1. Abundance of sterols in Dhcr7-/- and Dhcr24-/- cells.

Figure 2—figure supplement 1.

(A) Agarose gel electrophoresis showing amplification of relevant genomic DNA regions using primers that flank the two-guide CRISPR edits in the Dhcr7-/-, Dhcr24-/- and Lss-/- cell lines. A downward shift in the amplified band relative to the WT cell lines indicates successful editing. (B) Flow cytometry analysis (n > 4500 cells for each cell line) was used to measure staining of the plasma membrane by PFO*, a protein probe that binds accessible cholesterol (see Figure 5A and associated discussion). (C–F) Measurement of the indicated sterols in whole cell extracts by mass spectrometry (nd = not detected). Sterol measurements were normalized to a matched measurement of genomic DNA content in each sample to correct for differences in cell number (see Materials and methods). Each data point represents an independent plating of the indicated cell line (n = 4) and the height of the bar denotes the mean. Statistical significance in B-F was determined by the Mann-Whitney test; p-values are: (B) p-value<0.0001 (both comparisons), (C) p-value=0.0286 (both comparisons), (D) p-value=0.0286, (E) p-value=0.0286, and (F) WT vs Dhcr7-/-p value=0.0286; WT vs Dhcr24-/-p value=0.2.