Fig 2: PPAR? is involved in the upregulation of ABCA1 expression induced by allicin. (A) Small interfering RNA (siRNA) inhibited PPAR? expression. THP-1 macrophage-derived foam cells were transfected with scrambled or PPAR? siRNA. PPAR? expression was determined by western blot analysis and RT-qPCR. The results are expressed as the means ± SD from 3 independent experiments. *P<0.05 vs. controls. (B-D) Knockdown of PPAR? attenuated the allicin-induced upregulation of ABCA1 and LXRa expression. THP-macrophage-derived foam cells were transfected with 50 µmol/ml PPAR? siRNA or 10 mmol/l GW9662 and then incubated with 5 g/l allicin for 24 h. The expression of ABCA1 and LXRa was measured by RT-qPCR and western blot analysis. The results are expressed as the means ± SD from 3 independent experiments. *P<0.05 vs. allicin group. con, control; NG, negative siRNA; ABCA1, adenosine triphosphate (ATP)-binding cassette transporters A1; LXRa, liver X receptor a.

Fig 3: Knockdown ABCA1 attenuates the decreasing effects of allicin on lipid droplet accumulation in THP-1 macrophage-derived foam cells. THP-1 macrophage-derived foam cells were stimulated with PMA and ox-LDL (50 mg/l), and then incubated with 50 µmol/ml ABCA1 siRNA for 6 h, followed by treatment with 5 g/l allicin for 24 h. The cells were then stained with Oil Red O. Scale bar, 15 µm. Magnification, ×200. Black arrows indicate foam cells. ABCA1, adenosine triphosphate (ATP)-binding cassette transporters A1; ox-LDL, oxygenized low density lipoprotein; PMA, phorbol myristate acetate.

Fig 4: Allicin upregulates the expression of ABCA1 in THP-1 macrophage-derived foam cells. (A) THP-1 macrophage-derived foam cells were stimulated with PMA and ox-LDL (50 mg/l), and were then incubated with 50 µmol/l ABCA1 siRNA for 6 h, followed by treatment with 5 g/l allicin for 24 h. ABCA1 mRNA expression was analyzed by RT-qPCR. (B and C) THP-1 macrophage-derived foam cells were stimulated with PMA and ox-LDL (50 mg/l), and then treated with 5 g/l allicin for 24 h. The protein expression of ABCA1, LXRa and PPAR? was determined by western blot analysis. The results are representative of 3 independent experiments. aP<0.05 was used to compare the control group; bP<0.05 was used to compare the model group. con, control.ABCA1, adenosine triphosphate (ATP)-binding cassette transporters A1; LXRa, liver X receptor a; PPAR?, peroxisome proliferator-activated receptor ?; ox-LDL, oxygenized low density lipoprotein; PMA, phorbol myristate acetate.

Fig 5: ABCA1 and intracellular distribution of cholesterol are altered in SMA type I fibroblasts. (A) Representative images of ABCA1 immunostaining (green) in control (unaffected) and SMA type I fibroblasts. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (B) ABCA1 mRNA and GAPDH mRNA were checked in WCE and PMEF of control (unaffected) and SMA type I fibroblasts using semiquantitative RT-PCR. Panels are representative of three independent experiments. (C) Densitometric analysis of ABCA1 mRNA normalized to GAPDH mRNA in WCE and PMEF of control and SMA type I fibroblasts. The graph illustrates the means of three independent experiments. Error bars represent s.d. Asterisks indicate significant differences using unpaired t-test (*** p < 0.01). (D) Representative images visualizing free cholesterol via filipin staining in unstimulated and stimulated SMA type I fibroblasts compared to primary fibroblasts from a healthy individual. Scale bars, 10 µm. (E) Cholesterol staining intensity analysis in SMA type I-derived fibroblasts compared to primary fibroblasts from a healthy individual (Control), in both unstimulated and stimulated conditions. The graph illustrates the means of cholesterol fluorescence intensity determined by ImageJ software. Error bars represent s.d. Asterisks indicate significant differences using unpaired t-test (*** p < 0.01).