Fig 2: Downregulation of ICAM-1 expression (A, B, C, D, E) in pc3-GD3s cells. Expression levels of ICAM-1 mRNA were checked by RT-PCR (A) and real-time PCR (B). Expression of ICAM-1 confirmed by Western blot (C) and FACS analysis (D). A schematic representation of DNA constructions containing two different forms of the ICAM-1 promters linked to the luciferase reporter gene is shown. The values represent the mean ±S.D. for three independent experiments with triplicate measurements (E). Inhibition of AKT phosphorylation in pc3-GD3s cells (F). Total protein extracts from pc3 and pc3-GD3s cells were examined by Western blot analysis with anti-phospho-AKT, anti-AKT, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-p38 and anti-p38 antibodies. The nonspecific IgG antiserum has been used to block the cell reaction. GAPDH was included as an internal control. All experiments performed at least three times and we examined mean differences between groups by using the error bar graph procedure.

Fig 3: Effects of compound 2 on melanogenic proteins and mRNA levels in a-MSH- and IBMX-induced B16F10 cells. Cells were pre-incubated for 24 h and then stimulated with a-MSH (5 µM) and IBMX (200 µM) in the presence of compound 2 (5, 10, and 20 µM). The expression levels of tyrosinase, p-CREB, p-PKA, p-JNK, p-ERK, p-p38, and ß-actin were determined with Western blotting in the cytosolic fraction. ß-actin was used as the loading control (A,B,D,E). The expression levels of MITF and TFIIB were determined with Western blotting in the nuclear fraction. TFIIB was used as the loading control (C). Relative mRNA levels of the melanogenic genes MITF, Tyrosinase, Trp1, and Trp2 in the cells were quantified using real-time qPCR and normalized to the GAPDH mRNA level (F). The primer sets are shown in Table 1. Data are presented as the mean ± SEM of three independent experiments. ## p < 0.01 and ### p < 0.001 vs. control. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. a-MSH and IBMX-treated cells.

Fig 4: Influence of 5% CSE with or without JNK/p38MAPK signaling pathway inhibitors on the expression levels of Nrf2/MRP1 and the phosphorylation levels of JNK/p38 proteins in human bronchial epithelial cells. (A) Representative western blots and (B) semi-quantification of p-p38/t-p38, p-JNK/t-JNK, Nrf2 and MRP1. #P<0.05 vs. control group; *P<0.05 and **P<0.01 vs. CSE group. CSE, cigarette smoke extract; JNK, c-Jun N-terminal kinase; MAPK, mitogen activated protein kinase; Nrf2, nuclear erythroid factor 2; MRP1, multidrug resistance-associated protein 1; p-, phosphorylated; t-, total.

Fig 5: Effects of B19V-NS1, VP1 and VP1u on the phosphorylation of ERK and P38. (A) Protein expression levels of ERK, p-ERK, P38, and p-P38 in the left ventricle tissues of the mice receiving different treatments (PBS, VP1, NS1 or VP1u). (B) Relative levels of p-ERK and p-P38 are based on total protein expression. Similar results were obtained in three-repeated experiments. *P<0.05 vs. Control (mice treated with PBS). n=4 for each group. B19V, B19 virus; B19V-NS1, B19V-non-structural protein 1; VP1u, VP1-unique region; p-, phosphorylated.