Fig 5.

Silencing REST in HeLa cells inhibits expression, protein and activity of DHCR24 but does not alter DHCR7 mRNA or HMGR protein levels. All data presented as mean ± 95% CI. (a) qRT-PCR of REST, DHCR24 and a differently structured but essential sterol reductase, DHCR7, that appears to lack RE1 sequences. HeLa cells were transfected for four hours with pooled REST siRNA, then washed, grown in normal medium and harvested 2 days later (black bars). Controls (white bars, n=12) are cells transfected with random RNA. REST expression is reduced 10-fold and DHCR24 expression 2-fold but DHCR7 transcription remains unchanged; *P < 0.001, **P < 0.025 pre- vs. post-silencing, n=18, two-tailed t-test. (b) Relative REST, DHCR24 and HMGR protein levels (normalized to mean GAPDH) from cells incubated for 6 days with a stable lentivirus construct containing a REST targeting vector, shRNA (black bar), or an empty vector (white bar); *P < 0.0025, two-tailed t-test, n=6. HMGR protein is unchanged. (c) Ratios of two of the most abundant Δ²⁴-sterol precursors to their respective C24,25-saturated products after a 6 day incubation in lipoprotein deficient medium in cells treated with vectors containing (black bars) or empty of (white bars) REST silencing shRNA (lanosterol:24,25-dihydrolanosterol = 1.08 ± 0.23 vs. 3.36 ± 1.83, and desmosterol:cholesterol = 0.0055 ± 0.0029 vs. 0.022 ± 0.016, all n=9). *P = 0.068, **P = 0.028, two-tailed t-test.