Figure 2

Gene expression and enzyme activity analysis of HMG CoA reductase (HMGCR). (a) Quantification of HMGCR mRNA expression in human dermal fibroblasts from healthy controls (n = 4; white) and PXE patients (n = 4; black) grown for 24 h under different cell culture conditions (+10% FCS, +10% LPDS, −FCS). (b) Effect of siRNA-mediated knockdown on ABCC6 gene expression: fibroblasts transfected with a scramble siRNA-negative control (siNK, n = 4; white-striped); ABCC6-specific siRNA-treated cells (siABCC6, n = 4; black-shaded) grown for 24 h under different cell culture conditions (+10% FCS, +10% LPDS, −FCS). Expression levels are normalized to reference gene expressions (ACTB, GAPDH, β2M). Data are presented in arbitrary units as means with corresponding standard error. Control: PXE ratio/siABCC6: siNK ratio: ***p < 0.0001; *p < 0.03; Control 10% FCS: 10% LPDS, −FCS ratio/siNK 10% FCS: 10% LPDS, −FCS ratio: +++p < 0.0001; ++p < 0.004; PXE 10% FCS: 10% LPDS, −FCS ratio/siABCC6 10% FCS: 10% LPDS, -FCS ratio: ###p < 0.0001 (c, d) Enzyme activity of HMG CoA reductase in fibroblasts of healthy controls, PXE patients (c) and siRNA- treated cells (d) measured by UPLC-MS/MS. Data are presented in arbitrary units as means with corresponding standard error. Control: PXE ratio/siABCC6: siNK ratio: **p < 0.003; *p < 0.03; Control 10% FCS: 10% LPDS, -FCS ratio/siNK 10% FCS: 10% LPDS, −FCS ratio: +++p < 0.0001; ++p < 0.002; PXE 10% FCS: 10% LPDS, −FCS ratio/siABCC6 10% FCS: 10% LPDS, −FCS ratio: ###p < 0.0001; ##p < 0.009.