Figure 9.

SREBP1 positively regulates LGALS3 protein levels in human primary SMCs

Human primary aortic SMCs in full medium were transfected with a specific siRNA or scrambled control. The culture was then changed to fresh medium and continued until subconfluence. The cells were incubated with solvent control (0.25% BSA) or cholesterol at indicated concentrations for 72 h prior to harvest for assays. Because LGALS3 (and full-length SREBP1) protein levels peaked at 40 μg/mL cholesterol (see A), this concentration was used in the following experiments (see B–D). Data quantification (A–D): mean ± SEM, n ≥ 3 independent repeat experiments. Statistics: one-way ANOVA followed by Tukey test; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 between paired bars; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the basal condition (the first bar in each plot); non-significant comparisons are not labeled. (A) Upregulation of SREBP1 and LGALS3 proteins in cholesterol-loaded human SMCs. The colored bands are molecular weight markers. (B) SREBP1 silencing in human SMCs down-regulates LGALS3 protein. For KLF15, p = 0.031 and 0.057 when compared pairwise using Student’s t test. (C) Pre-treatment of human SMCs with JQ1 (0.1 or 0.5 μM) inhibits cholesterol-induced SREBP1 and LGALS3 protein production. Cells were incubated with JQ1 (0.1 or 0.5 μM) or vehicle (DMSO) for 1 h prior to cholesterol loading. (D) LGALS3 negatively regulates MRTF-A protein levels in human SMCs.