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. 2022 May 18;28:892–909. doi: 10.1016/j.omtn.2022.05.028

Figure 1.

Figure 1

SREBP1 positively regulates LGALS3 expression in mouse and rat SMCs

The SMCs used in experiments were mouse MOVAS cells unless otherwise specified (i.e., rat primary aortic SMCs in C). For loss or gain of function, SMCs were transduced with lentivirus for 24 h to express a specific shRNA or scrambled control, or to express a gene or empty vector (EVec) control. The culture was then changed to fresh medium and continued until subconfluence prior to cholesterol loading. Cells cultured in full medium were incubated with solvent control (0.25% BSA) or cholesterol (Chol; in a Chol-methyl-β-cyclodextrin soluble form) at indicated concentrations for 72 h prior to harvest for various assays. Data quantification for western blots: mean ± SEM, n ≥ 3 independent repeat experiments. Data quantification for quantitative real-time PCR and ChIP-qPCR: mean ± SD, n = 3 replicates; presented is one of two similar 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. Note: Srebf1 produces two highly homologous mRNA variants and protein variants (SREBP1a and SREBP1c); the primers and antibody used here do not distinguish between these two variants. (A) Upregulation of LGALS3 and SREBP1 proteins (western blot) and mRNAs (quantitative real-time PCR) by increasing concentrations of cholesterol. (B) SREBP1 silencing in MOVAS cells reduces LGALS3 protein and mRNA expression. (C) SREBP1 silencing in primary rat aortic SMCs reduces LGALS3 protein. (D) SREBP1 overexpression in MOVAS cells increases LGALS3 protein. (E) ChIP-qPCR showing SREBP1 binding at a proximal site of the Lgals3 promoter. S1, S2, and S3 denote three software-predicted SREBP1-binding sequences at different Lgals3 promoter sites. ChIP was performed using an antibody specific for endogenous SREBP1, after MOVAS cells were treated for the indicated hours. r.u., relative unit; SREBP1Nterm, the N-terminal half molecule, which is the active form of the SREBP1 transcription factor.