Fig. A, Quantification of ZIPK mRNA levels in CASMC transfected with ZIPK or ROCK1 siRNAs.

CASMCs were transfected with siRNA to ZIPK or ROCK1 or with negative control siRNA. Cells were lysed 48 h later for qRT-PCR to quantify ZIPK mRNA levels. Values represent means ± SEM (n = 9). *significantly different from control (p < 0.001). Fig. B, Effect of ROCK1 and ZIPK knockdown on myosin phosphorylation. Representative western blots of control, ZIPK- and ROCK1-knockdown UASMC with anti-2P-LC20 (antibody recognizing LC20 only when phosphorylated at Thr18 and Ser19) and GAPDH as loading control. See Table 12 for cumulative quantitative data. Table A, ROCK1 and ZIPK knockdown in UASMC at the protein level. UASMC were transfected with siRNAs targeting ROCK1 or ZIPK or with negative control siRNA as described in the Materials and Methods section. The efficiency of knockdown at the protein level was determined by western blotting. ROCK1 and ZIPK signals were normalized to GAPDH. Values are expressed relative to levels in control cells (means ± SD, n = 11 for ROCK1 knockdown and n = 8 for ZIPK knockdown). *significantly different from control (p < 0.001). Table B, Genes whose expression is altered by ROCK1 knockdown. Table C, Genes whose expression is altered by ZIPK knockdown. Table D, Effects of ZIPK and ROCK1 knockdown on cytokine secretion. A Human Custom Multi-Analyte ELISArray kit (CELISA-CMEH0590A) was purchased from Qiagen. The indicated cytokines were assayed in the medium of CASMC transfected with control siRNA (Control) and CASMC transfected with siRNA to ZIPK (ZIPK knockdown) or ROCK1 (ROCK1 knockdown). Positive controls provided with the kit verified the viability of the assay for each cytokine. Negative controls indicated that, of the 6 cytokines listed, IL-1α, MCP1 and GROα were secreted at detectable levels. Values indicate absorbance at 450 nm ± S.E.M. (n = 4). *p < 0.05 compared to Control.
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