Figure 2
From: Regulation of Cholesterol Homeostasis by a Novel Long Non-coding RNA LASER
LASER regulate cholesterol metabolism through HNF-1α and PCSK9. (A) The expression of LASER was quantified by qRT-PCR in HepG2 cells incubated for 48 hrs with siRNA against LASER or scramble control (n = 6, *P < 0.05 versus control). (B,C) Effect of LASER siRNA on intracellular cholesterol level in HepG2 cells. After LASER knocked-down by siRNA, the intracellular cholesterol level was measured by Amplex red assay (B) (n = 6, *P < 0.05 versus control), or stained with filipin and observed by confocal laser-scanning microscopy (C). (D) Hierarchical clustering analyses of deregulated protein-coding genes (>1.2 folds change) after LASER knockdown in HepG2 cells. Up- and down-regulated genes are represented by red and green respectively (n = 3). (E) Volcano plot illustrating significant differential gene expression (mRNAs) changes (red marks) attributed to LASER knock-down. −Log10 (Benjamini–Hochberg-corrected p value) p < 0.05; Log2 (fold change) >1.2-fold (n = 3). (F) Pathway analysis indicating the number of deregulated genes (>1.2 folds change) after LASER knock-down. (G) Some of the key genes implicated in cell cholesterol metabolism were verified by qRT-PCR after LASER knocked-down. Gene expression were normalized to GAPDH (n = 6, *P < 0.05 versus control). (H,I) The concentration of PCSK9 was detected using ELISA methods in both cell lysate (H) and cell culture supernatants (I) of HepG2 cells treated with LASER siRNAs (50 nM) or scramble control (n = 6, *P < 0.05 versus control). (J) The protein levels of HNF-1α, PCSK9, LDLR and GAPDH were determined by western blotting after siRNA (50 nM) against LASER or scramble control (n = 6, *P < 0.05 versus control). (K) HepG2 cells were treated with different concentrations of berberine (0, 5, 10, 15 µg/ml) with or without LASER siRNA (50 nM) for 24 hrs. The expression levels of PCSK9 mRNAs were determined by qRT-PCR (n = 6, *P < 0.05 versus control, N.S: not significant).
