Figure 1.
The up-regulation of K17 expression in IL-22-induced keratinocytes.
(A) The real-time PCR analysis of K17 mRNA levels. Data are expressed as 2−ΔΔCT relative to untreated HaCaT cells. (B) The ELISA analysis of K17 expression. (C) The Western blot analysis of K17 protein expression. (D) Immunofluorescence was performed on HaCaT cells to measure K17 expression. DAPI staining for nuclei is in blue. The scale bars represent 30 µm. The blank group is untreated HaCaT cells. Results represent means±SEM from three independent experiments. *P<0.05 was considered significant for the IL-22 treated group versus blank.
Figure 2.
The activation of STAT3 and ERK1/2 signaling pathways in IL-22-treated HaCaT cells.
HaCaT cells were treated with IL-22 and the expression of STAT3, ERK1/2, phospho-STAT3 or phospho-ERK1/2 was tested with corresponding antibodies. (A) The Western blot analysis of the activation of phospho-STAT3 and phospho-ERK1/2 in IL-22-treated HaCaT cells. (B) Immunofluorescence staining of phospho-STAT3 and phospho-ERK1/2 in IL-22-treated-HaCaT cells at different time points. Note that stronger signals were observed in the cultures at 15 min, 30 min or 60 min following IL-22 treatment. DAPI staining for nuclei is in blue. The scale bars represent 30 µm. The blank group is untreated HaCaT cells.
Figure 3.
Inhibition of STAT3 and ERK1/2 signaling pathways partially suppresses the effect of IL-22 on K17 expression.
(A) An examination of the inhibitory effect of piceatannol and on PD98059K17 mRNA levels with real-time PCR analysis. (B) The Western blot analysis of the inhibitory effect of piceatannol and PD98059 on K17 protein expression. (C) Immunofluorescence staining of K17 expression in piceatannol and PD98059-treated, IL-22 stimulated HaCaT cells, or untreated HaCaT cells. DAPI staining for nuclei is in blue. The scale bars represent 30 µm. (D) An examination of the inhibitory effect of STAT3 and ERK1/2 siRNA on K17 mRNA levels with real-time PCR analysis. (E) The Western blot analysis of the inhibitory effect of STAT3 and ERK1/2 siRNA on K17 protein expression. The blank group is untreated HaCaT cells. *P<0.05 was considered significant.
Figure 4.
The epidermal proliferation and up-regulation of K17 induced by IL-22 in the mouse skin.
(A) The real-time PCR analysis of K17 mRNA levels. The results represent means±SEM from three independent experiments. (B) H&E-stained sections of PBS- or IL-22-injected ears after daily injection for seven days. The epidermal thickness was measured. Data are from 2 experiments with 5 mice per group. (C) Histological and immunohistochemical analysis of K17 stimulated or not with 20 ng/ml IL-22. 4 µm vertical sections were reacted with rabbit anti-K17 then photographed under a microscope (magnification×400). (D) Frozen sections of ears from PBS-injected or IL-22-injected mice stained with rabbit anti-mouse K17 followed by FITC-conjugated goat anti-rabbit IgG (green). DAPI staining for nuclei is blue. *P<0.05 was considered significant for the IL-22 injected mouse group versus PBS control.
Figure 5.
The synergism of IL-22, IL-17A and IFN-γ in inducing K17 expression.
HaCaT were treated with IL-22(25 ng/ml), IL-17A (100 U/ml) and IFN-γ (100 U/ml) alone or in combination. (A) The real-time PCR analysis of K17 mRNA levels after 24 h; Data are expressed as 2−ΔΔCT relative to untreated HaCaT cells. (B) The Western blot analysis of K17 protein expression. The blank group is untreated HaCaT cells. Results represent means±SEM from three independent experiments. *P<0.05 was considered significant.