Figure 1

Characterization of vascular smooth muscle cells (VSMCs). (A) Generation of VSMCs overexpressing PCSK9 by using a pBMN-IRES-puromycin vector carrying human PCSK9 coding sequence. FLAG peptide has the following sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys; PURO stands for puromycin; IRES stands for internal ribosome entry site; LTR stands for long terminal repeat. Protein expression of FLAG was assessed by WB analysis. β-actin was used as a housekeeping protein. Representative image of three independent experiments. (B) Gene (quantitative PCR) and protein expression (WB) of PCSK9 in VSMCs. 36B4 was used as a reference gene and β-Actin was used as a housekeeping protein. For WB, the reported blot is representative of three independent experiments. (C) Data obtained from the ELISA assay are expressed as % of PCSK9 released in the cell culture medium of VSMCs^PCSK9 compared to VSMCs^WT; the results were normalized for the total protein content. n = 6 per group. (D) Proliferative assay of VSMCs (cell counting). n = 6 per group. (E) VSMCs were cultured in standard conditions for seven days and the morphology was evaluated by optical microscopy [17]. (F) Gene expression of VSMC markers(Acta2 and Calponin). β-Actin was used as a reference gene. n = 6 per group. (G) Gene ontology enrichment analysis relative to proteins with a significantly higher expression in VSMCs^PCSK9. Red bar represents VSMCs^PCSK9 and white bar represents VSMCs^WT. Results are expressed relative to the normal control and as mean ± SEM. Differences between groups have been assessed by t-test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control. Acta2, alpha-actin-2; PCSK9, proprotein convertase subtilisin/kexin type 9; VSMCs, vascular smooth muscle cell; VSMCs^PCSK9, vascular smooth muscle cells overexpressing PCSK9; VSMCs^WT, vascular smooth muscle cells wildtype; WB, Western blot.