Figure 1.
Effects of TRAIL on lipid uptake and foam cell formation in macrophages.
(A) Fluorescence images showing the effects of TRAIL on DiI-Ac-LDL (red) uptake in RAW264.7 cells. Cells were pretreated with TRAIL (10 ng ml−1) for 24 hr, then loaded with DiI-Ac-LDL for different time as indicated. (B) Quantitative data of the fluorescence intensity in (A). (C & D) Effects of TRAIL (10 ng ml−1 for 24 hr) on DiI-Ac-LDL uptake in PMA-differentiated human THP-1 cells. (E) Effects of TRAIL on ox-LDL-triggered foam cell formation in RAW264.7 and THP-1 cells as detected by Oil Red O staining (red color, arrows). * P<0.05 and ** P<0.01 vs control, unpaired t-test or one-way ANOVA followed by Newman-Keuls test, n = 3.
Figure 2.
Effects of TRAIL on foam cell formation in wild type (DR5+/+) and DR5-deficient (DR5−/−) peritoneal macrophages.
Cells were treated with TRAIL at 10−1 for 24 hr, and then loaded with ox-LDL (80 µg ml−1) for 48 hr. Internalized LDL was detected with Oil Red O staining (red color).
Figure 3.
Effects of varying concentrations of TRAIL on expressions of different scavenger receptors in RAW264.7 cells.
Cells were treated with TRAIL for 24(A to D) Quantitative real-time PCR results of mRNA expression of scavenger receptors. (E & F) Time course of SR-AI and SR-BI expression in cells treated with TRAIL (10 ng ml−1). (G) Western blot showing the effect of TRAIL on SR-AI protein expression (n = 3 independent experiments). The PCR results are expressed as fold of control (Con). *P<0.05 vs Con, **P<0.01 vs Con, one-way ANOVA, n = 3–6.
Figure 4.
Effects of varying concentrations of TRAIL on expressions of scavenger receptors in THP-1 cells.
(A to D) Quantitative real-time PCR results of mRNA expression of scavenger receptors. (E) Western blot showing the effect of TRAIL on SR-AI protein expression (n = 3 independent experiments). The PCR results are expressed as fold of control (Con). * P<0.05 vs Con, one-way ANOVA, n = 3–6.
Figure 5.
TRAIL-stimulated scavenger receptor expressions were blocked by TRAIL neutralizing antibody or DR5 deficiency.
(A to D) Blockade of the effects of TRAIL on SR-AI and SR-BI expression with a TRAIL neutralizing antibody (NAb) (5 µg ml−1) in RAW264.7 (A & B) and THP-1 cells (C & D). Bovine serum albumin (BSA) in saline buffer was used as vehicle control. (E & F) The effects of TRAIL on SR-AI and SR-BI expression were abolished in DR5-deficient peritoneal macrophages. * P<0.05 vs control (Con), one-way ANOVA or unpaired t-test, n = 3–4.
Figure 6.
Effects of blockade of SR-AI and SR-BI functions on TRAIL-stimulated DiI-Ac-LDL uptake in macrophages.
(A) Effects of the SR-AI inhibitor poly(I:C) (1 µM) and the SR-BI inhibitor BLT-1 (5 µM) on TRAIL-stimulated DiI-Ac-LDL uptake in RAW264.7 cells. (B) Western blot showing the effect of siRNA on protein expression of SR-AI in RAW264.7 cells. Cells were incubated with control or SR-AI siRNA (all at 200 nM) for 48 hr. (C & D) Quantitative data showing the knocking down effects of siRNA on SR-AI mRNA and protein levels measured by real-time PCR or western blot (E) SR-AI siRNA blocked the stimulating effect of TRAIL on DiI-Ac-LDL uptake in RAW264.7 cells. (F) Effects of poly(I:C) and BLT-1 on TRAIL-stimulated DiI-Ac-LDL uptake in THP-1 cells. * P<0.05 vs control (Con), one-way ANOVA or unpaired t-test, n = 3–4.
Figure 7.
Role of MAPKs in TRAIL-induced SR-AI expression.
(A) Western blot showing the effects of TRAIL (10 ng ml−1) on phosphorylation of ERK1/2, p38 and JNK MAPKs in RAW264.7 cells. TNF-α (20 ng ml−1) was used as a positive control. The total levels of FADD or TRAF2 were not changed. (B) Effects of the ERK pathway inhibitor U0126 (1 µM), p38 inhibitor SB202190 (1 µM) and JNK Inhibitor II (1 µM) on TRAIL-stimulated SR-AI expression. The results are expressed as fold of control (Con). * P<0.05 vs Con, one-way ANOVA, n = 3.
Figure 8.
Effects of TRAIL on apoptosis of macrophage and vascular smooth muscle cells (VSMCs).
(A) Representative images of TUNEL labeling showing apoptotic cells (arrows) in control and TRAIL-treated (100 ng ml−1 for 24 hr) RAW264.7 cells. (B) Quantitative analysis of TUNEL-positive RAW264.7 cells after treatment with TRAIL (10, 100 and 400 ng ml−1) for various time as indicated. (C) Quantitative analysis of TUNEL-positive cells in VSMCs after treatment with TRAIL for different time. Data are % of total cells averaged from triplicate experiments.
Figure 9.
TRAIL-stimulated lipid uptake and SR-AI expression were not dependent on cell apoptosis.
(A) Effect of co-treatment with the pan-caspase inhibitor z-VAD-fmk (1 µM) on TRAIL (10 ng ml−1)-stimulated DiI-Ac-LDL uptake in RAW264.7 cells. (B) Quantitative data of the fluorescence intensity in A. (C) Effect of z-VAD-fmk on TRAIL-stimulated SR-AI mRNA expression. (D) Effect of staurosporine (STS, 1 µM)-induced apoptosis on SR-AI expression. Cells were treated with STS for 1 hr and then incubated in fresh medium for additional 24 hr. * P<0.05 vs control (Con), one-way ANOVA, n = 3.