Figure 1.
STS induces keratinocyte migration and STC1 mRNA expression.
(A) In the cell spreading assay, e-lam formation on fibronectin-coated plate was induced by increasing doses of STS treatment (0, 2.5, 5, 10 nM) for 24 h. The cell images were captured under magnification of 200x (top). (B) Cell migration through the transwell was induced by 5 nM STS for 24 h and the cell images were captured under 100x magnification (top). (C) Western blot analysis demonstrated the increase of FAK phosphorylation (pY397) upon 5 nM STS treatment from 10–360 s. (D) Cell migration was increased upon STS treatment at 24 h, but was blocked by 2 µM PF573228. Cell images were captured under 100x magnification (top). (E) STC1 mRNA and protein expressions were induced by increasing doses of STS treatment (0, 2.5, 5, 10 nM). (F) STS-induced STC1 mRNA expression was not significantly abolished by PF573228 at 24 h. Asterisks (***) denote p<0.0001, (**) denote p<0.005 and (*) denote p<0.01 as compared to the respective control treatment.
Figure 2.
STS-induced migration is mediated by intracellular Ca2+ influx.
(A) STS treatment increased [Ca2+]i as measured using Ca2+ detection dye, Fura 2-AM. (B) The number of migrated cells was significantly reduced by 10 µM BAPTA/AM at 24 h (vs Ctrl) and the cell images were captured under 100x magnification (top). (C) STS-induced STC1 mRNA was further induced by 1 µM ionomycin but was downregulated by 10 µM BAPTA/AM at 24 h of the cotreatments. Asterisks (*) denote p<0.05 as compared to the Ctrl.
Figure 3.
Akt-GSK3β signaling mediates the keratinocyte migration process.
(A) The inhibitory effects of 30 mM LiCl and 10 µM SH6 on GSK3β and Akt-signaling respectively, were determined by probing the phosphorylated and total forms of GSK3β and Akt. The band intensities were measured and the ratios of phosphorylated/total form were plotted. Bars with the same letter are not significantly different according to the results of one-way ANOVA followed by Duncan’s multiple range tests (p<0.05). STS-induced (B) e-lam formation and (C) cell migration were suppressed by LiCl but were induced by SH6. The cell images were captured under 200x and 100x magnification for e-lam formation and cell migration, respectively (top). (D) STS-induced STC1 mRNA expression was inhibited by LiCl, but was increased by SH6 (vs STS alone). Asterisks (*) denote p<0.05 as compared to their corresponding control.
Figure 4.
STC1 knockdown hinders STS-mediated re-epithelialization process.
(A) After the transfection of STC1 siRNA for 48 h, the knockdown efficiency was examined by real-time PCR. In STC1 siRNA-transfected cells, STS-induced (B) cell migration and (C) e-lam formation on the fibronectin-coated plates were significantly reduced as compared to the NS siRNA-transfected cells. The cell images were captured for e-lam formation (200x magnification) and cell migration (100x magnification) (top). Under the STS+LiCl or STS+SH6 cotreatment, (D) the number of migrated cells and (E) e-lam formation on fibronectin-coated plate were compared between the STC1 siRNA-transfected cells and the NS siRNA-transfected cells. The knockdown of STC1 was found to inhibit cell migration induced by the STS + LiCl/SH6 cotreatments. The synergistic effect of STS/SH6 on the increase of e-lam formation was significantly reduced in the STC1 siRNA transfected cells. Asterisks (**) denote p<0.01 and (*) denote p<0.05 as compared to their corresponding NS siRNA-transfected cells.
Figure 5.
STC1 enhances STS-mediated re-epithelialization process.
(A) After transient transfection of V5-tagged STC1/pLenti (STC1/pLenti) and empty vector control (pLenti) into the HaCaT for 24 h, the cells were treated with 5 nM STS for 24 h and the protein expression levels of V5, GSK3β and Akt were determined using western blotting. The band intensities were measured and the ratios of the respective phosphorylated/total proteins were plotted. STC1 overexpression was found to have synergistic effect on STS-inhibited phosphorylation of Akt. STS-induced (B) e-lam formation on fibronectin-coated plate and (C) cell migration were synergistically induced by the transient overexpression of STC1 (vs STS-treated pLenti). The cell images were captured for e-lam formation (200x magnification) and cell migration (100x magnification) (top). (D) In the scratched wound healing assay, the scratched wound was closed significantly more rapidly in the cells maintained in the conditioned medium (CM) containing overexpressed STC1 protein (CM-STC1) than the control medium (CM Ctrl). The cell images were captured under 100x magnification. The levels of STC1 proteins in the conditioned media (CM-Ctrl and CM-STC1) were determined from day1 to day3 using western blotting. (top); CM-Ctrl depicts as C and CM-STC1 depicts as T. Asterisks (**) denote p<0.01 and (*) denote p<0.05 as compared to STS-treated pLenti-transfected cells.