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. 2011 Mar 25;286(20):17618–17630. doi: 10.1074/jbc.M111.226563

FIGURE 1.

FIGURE 1.

Involvement of CD44 in TGFβ1-driven proliferation in dermal fibroblasts. A, CD44 mRNA expression. Dermal fibroblasts were transfected with CD44 or negative control (scrambled) siRNA and incubated for 24 h. To confirm CD44 knockdown, mRNA extraction was carried out, and CD44 mRNA expression was assessed by RT-QPCR. Ribosomal RNA was used as an endogenous control, and gene expression was assessed relative to the scrambled samples. The comparative CT method was used for relative quantification of gene expression, and the results are expressed as box plots. For each box plot, the box represents 50% of the values (the 25th and 75th percentiles), with the bars presenting the highest and lowest values. Statistical analysis was performed using the Wilcoxon signed-rank test, and the results are representative of three experiments. B, alamarBlueTM proliferation assay. In parallel experiments, the effect of CD44 knockdown on TGF-β1-driven proliferation in dermal fibroblasts was assessed using an alamarBlueTM assay (as described under “Experimental Procedures”). As above, the siRNAs were incubated with the cells in serum-free medium for 24 h. Subsequently, 10 ng/ml TGF-β1 was incubated with the cells for a further 24 h prior to analysis. The results are expressed as the means ± S.E. (error bars) for three experiments. C, [3H]thymidine proliferation assay. The effect of CD44 knockdown on TGF-β1 driven proliferation in dermal fibroblasts was also assessed using a [3H]thymidine assay (as described in “Experimental Procedures”). As above, the siRNAs were incubated with the cells in serum-free medium for 24 h. Subsequently, 10 ng/ml TGF-β1 was incubated with the cells for a further 24 h prior to analysis. The results are expressed as box plots. For each box plot, the box represents 50% of the values (the 25th and 75th percentiles), with the bars presenting the highest and lowest values. Statistical analysis was performed using the Friedman test followed by the Wilcoxon signed-rank test, and statistical significance was taken as p < 0.05. The results are representative of three experiments.