Figure 6.

Down-regulation of HAS2 by shRNA. Lentiviral transfection with shRNA targeting HAS2 (shHAS2) and empty control vector (pCL1) was performed in human skin fibroblasts. A: Five days after infection, analysis of HAS2 mRNA expression was performed using real-time RT-PCR. B: In parallel, HA secretion was determined by enzyme-linked immunosorbent assay, normalized to total protein and expressed as percentage of pCL1-transfected cells. C: Subsequently, on day 6 after infection, the migration toward 10% FCS was determined using a modified Boyden-chamber microchemotaxis assay on collagen type-1-coated membranes. D: [³H]Thymidine incorporation was determined on day 6 after transfection using 10% FCS as mitogenic stimulus. The data were normalized to total protein and represent the fold increase over starved controls. Data are means ± SEM of n = 4 independent experiments each performed in triplicates. P values were calculated by t-test; *P values <0.05.