(
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.