Fig 1.
Effects of NTP treatment on cell migration, cell death, cell viability and protein expression in KFs and NFs.
(A) Scratch wound healing assay after NTP treatment on NF and KF. Wound healing was documented by photography. (B) Annexin-V and propidium iodide staining in NFs and KFs. (C) MTT assay for cell viability of NFs and KFs. (D) Western blotting analysis for T-EGFR, Type I collagen, p-STAT3, T-STAT3, p-AKT, T-AKT, p-FAK, T-FAK, vimentin, p-ERK, T-ERK in NFs and KFs after NTP treatment. (E)Immunofluorescent staining of NFs and KFs was performed using p-STAT3 and F-actin antibody. DAPI was used for nuclei counter staining. After NTP exposures for 10, 30, or 60 s and after the control gas treatment.(Green = p-STAT3, RED = phalloidin, F-actin, Blue = DAPI). Data represent mean±standard deviation of three independent experiments. Each figure was representative of three experiments with triplicates. Scale bar = 100 μm. *P < .05, **P < .01 and ***P < .001.
Fig 2.
Comparisons of protein expression and protein phosphorylation in NFs and KFs.
(A) The expression levels of p-EGFR, T-EGFR, T-STAT3, p-STAT3, and Type I collagen in NS and KF tissues were compared. Quantitative analysis of expression levels were made in NS (5 cases) and KF (5 cases). The expression of is EGFR, p-STAT3 and Type I collagen increased in KF tissues compared with NS. (B) Haematoxylin-eosin staining and immunohistochemical staining with antibodies against p-STAT3, T-EGFR, and Type I collagen and III were performed in paraffin sections of normal and keloid skin tissue specimens from patients. Data represent mean±standard deviation of three independent experiments. Each figure was representative of three experiments with triplicates. Scale bar = 100 μm. *P < .05, **P < .01 and ***P < .001.
Fig 3.
Effects of EGFR and STAT3 specific siRNAs on NFs and KFs.
(A) Migration of NFs and KFs was assessed by the wound healing assay. Cell migration of NFs and KFs was lower after 24 and 48h treatments with EGFR-specific rather than control siRNA. (B) STAT3 siRNA had a higher inhibitory effect on cell migration in NFs and KFs than control siRNA after 24 and 48h treatments. (C) Western blotting for T-EGFR, p-EGFR, p-AKT, T-AKT, p-ERK, T-ERK, p-STAT3, T-STAT3, Type I collagen, vimentin, and α-tubulin in NFs and KFs after treatment with EGFR and STAT3 specific siRNAs. Data represent mean±standard deviation of three independent experiments. Each figure was representative of three experiments with triplicates. Scale bar = 100 μm. *P < .05, **P < .01 and ***P < .001.
Fig 4.
Collagen production after NTP treatment in NFs and KFs.
(A) Reductions in Type I collagen, Type III collagen, and TGF-β mRNA expression evaluated by RT-PCR in KFs that were exposed to NTP for 10, 30, and 60 s as well as to control gas treatment. (B) NTP reduced the expression of Type I collagen in KFs as revealed by the immunocytochemistry and Sirus Red stain. Immunofluorescent and Sirus Red staining for Type I collagen were observed in NFs and KFs after treatments with gas or NTP. (C) Total soluble collagen were measured by Sircol collagen assay kit after treatment with NTP in NF and KF. (D) Collagen production levels were measured after combination treatment with NTP and STAT3 siRNA in NF and KF. Data represent mean±standard deviation of three independent experiments. Each figure was representative of three experiments with triplicates. Scale bar = 100 μm. *P < .05, **P < .01 and ***P < .001.