Figure 5: Endothelial-expressing Zdhhcs and importance of DHHC5/21 in endothelial dysfunction.

(a) Representative images from qualitative PCR analyses of 24 known murine Zdhhcs in primary MLMVECs that confirmed the expression of 11 Zdhhcs. cDNA template of each gene was used as a positive control. (b) Knockdown efficiency of the 11 Zdhhcs expressed in endothelial cells verified by RT-PCR, *P<0.05 versus control siRNA. (c,d) The effects of individual Zdhhc knockdown on thrombin-induced endothelial barrier dysfunction indicated by TER. (c) Representative TER tracings of Zdhhc5 and Zdhhc21 knockdown in response to thrombin (10 U ml⁻¹). Solid line tracing represents the mean resistance, and shadow represents±s.e.m. n=number of measurement; value in parentheses indicates number of independent experiments. (d) Quantification of peak TER reduction upon thrombin treatment in each Zdhhc knockdown group. Values were normalized to TER with control siRNA. Bar graph represents mean±s.e.m. from three independent experiments. *P<0.05 versus control siRNA. (e,f) The effects of individual Zdhhc knockdown on IL-1β-induced ICAM-1 expression on EC surface. MLMVECs in a 96-well plate were stimulated with IL-1β (100 ng ml⁻¹, 4 h). ICAM-1 surface expression was determined by on-cell western assay using Odyssey CLx Infrared Imaging System. (e) Representative images. CellTag staining was used for cell number normalization. Arrow indicates decreased ICAM-1 expression. (f) Quantification of ICAM-1 expression in Zdhhc knockdown ECs upon IL-1β stimulation. Results represent mean±s.e.m. from three independent experiments. *P<0.05 versus control siRNA.