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Figure 4: AQP3 and FLG gene expression analysis in HaCaT cells treated with HSEE and HSEE effect on ex vivo skin explants. (a) HaCaT cells were seeded at concentration of 1.5 × 105 cells in 35 mm dishes. After 24 h, cells were treated for 6 h with HSEE at the indicated concentrations. Total RNA was prepared and reverse-transcribed. Obtained cDNAs were used to evaluate AQP3 and FLG gene expression by performing a semiquantitative RT-PCR. Retinoic acid at concentration of 1 μM was used as positive control. The values were normalized to rRNA 18S. Each column value represents the average of four experiments and error bars indicate standard deviations. value < 0.05 is represented by ; value < 0.01 is represented by . value < 0.001 is represented by . (b) Representative experiment showing the effect of HSEE on ex vivo skin explants. The samples were wounded and treated with HSEE and TGFβ as indicated. After 3 days, the explants were processed to produce sections, which were then stained for morphology analysis. (c) Fibronectin was detected by immunostaining using a monoclonal primary antibody (Santa Cruz Biotechnology) and FITC-conjugated secondary antibody (green). The nuclei were poststained with propidium iodide (red). The length of the newly generated epidermis is indicated by the dotted lines. The leading edge is indicated by arrows.