Figure 5. ORP2 silencing down-regulates genes required for cholesterol mobilization.

(A) Total RNA from control and Bt₂cAMP-treated H295R wild type and ORP2^kd cells was isolated using RNeasy Mini Kit, standardized to 10 ng/µl, and amplified in triplicate using a One-Step SYBR Green RT-PCR Kit for HSL, LDLR, SCARB1 and StAR. Gene expression was normalized to β-actin mRNA content and calculated using the ΔΔ cycle threshold (ΔΔCT) method. Data are graphed as fold change over untreated control and represent the mean ± SEM of five separate experiments, each performed in triplicate. Asterisks and carats denote statistically significant differences when compared to wild type control and wild type Bt₂cAMP-treated, respectively. (B) Wild type and ORP2^kd H295R cells were treated with Bt₂cAMP (0.4 mM) for 48 h, and whole cell lysates harvested and separated by SDS-PAGE followed by western blotting using antibodies against StAR, LDLR, SRB1, HSL, and GAPDH. Shown are representative blots, where the protein expression of each gene was analyzed in at least duplicate. Data graphed in B represent the mean ± SEM of densitometric analysis of protein expression from four independent experiments. (C) Cholesterol, 25-hydroxycholesterol, 22-hydroxycholesterol, and 7-ketocholesterol levels in wild type and ORP2^kd cells were quantified by mass spectrometry as described in the Materials and Methods. Data are expressed as pmol per 5 × 10⁶ million cells.