Figure 4.

Inhibitory effect of the phosphorylation of the JNK, p38 MAPK, and ERK1/2, and the transcription factors c-Jun and c-Fos, in UV-B-exposed and NK-CdM-treated NHDFs. After being serum-starved for 24 h, NHDFs were irradiated with UV-B (30 mJ/cm²), and further incubated with NK-CdM at the indicated concentrations for 48 h. Total cell protein extracts were prepared and separated by electrophoresis on SDS-PAGE gels followed by a western blot analysis with primary antibodies capable of detecting (A) phospho-JNK, -p38 MAPK, and -ERK1/2 and (B) phospho-c-Jun, -c-Fos. β-actin was used as the loading control. (C) Equal protein loading was verified by analyzing β-actin levels. (D) The antioxidant activity of NK-CdM, at the indicated concentration, was measured using an Antioxidant Assay kit. (E) NHDFs were treated with 10 μM of the ERK1/2 inhibitor PD, 10 μM of the JNK inhibitor SP, 10 μM of the p38 MAPK inhibitor SB, or 10 μM of the PI3K inhibitor LY and then irradiated. The levels of procollagen I and type I collagen at 48 h after irradiation were measured by western blot analysis. (F) The bar graphs (mean ± standard error of the mean; n=3) represent a quantitative densitometric analysis of the bands. ^#P<0.05 and ^###P<0.001 vs. the Normal (non-irradiated control). ^**P<0.01 and ^***P<0.001 vs. the UV-B-irradiated control. ^$P<0.05, ^$$P<0.01 and ^$$$P<0.001 vs. the UV-B irradiated and 1.25% NK-CdM treated group. MMP, matrix metalloproteinase; ERK, extracellular signal regulated kinase; NK-CdM, natural killer cell conditioned medium; NHDF, neonatal human dermal fibroblasts; UV, ultraviolet; MAPK, mitogen associated protein kinase; PI3K, phosphatidylinositol 3 kinase; SP, SP600125; LY, LY294002; SB, SB203580; PD, PD98059; phosphor, phosphorylated; JNK, c-Jun N terminal kinase.