1. CX-659S, a Diaminouracil Derivative, Indirectly Inhibits the Function of Langerhans Cells by Blocking the MEK1/2-Erk1/2 Pathway in Keratinocytes 
Hiroshi Uchi, Tetsuya Koga, Kazunori Urabe, Yoichi Moroi, Masutaka Furue 
Journal of Investigative Dermatology 
Volume 120, Issue 6, Pages (June 2003)
DOI: /j x
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Effect of CX-659S on the expression of surface molecules on LC. Suspensions of epidermal cells were treated with increasing concentrations of CX-659S (0–500 μM) for 72 h in the presence (white circles) or absence (black circles) of GM-CSF (20 ng per ml). The expressions of surface molecules on Ia-positive LC were analyzed. The mean fluorescence intensity of each molecule was measured using a flow cytometer, and percentages of mean fluorescence intensity levels compared with those of respective control conditions (CX-659S 0 μM) were calculated. Data are means±SEM of four independent experiments. *p<0.05 compared with respective control conditions.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Effect of CX-659S on production of IL-2 from allogeneic T cells. (A) Suspensions of epidermal cells were treated with CX-659S (0–500 μM) for 24 h in the presence (white circles) or absence (black circles) of GM-CSF (20 ng per ml) and then cells were washed out. CX-659S-treated epidermal cells were cocultured with allogeneic T cells for 72 h and the amount of IL-2 in each culture supernatant was measured. Data are means±SEM of three independent experiments. *p<0.05. (B) T cells were also stimulated with PMA and ionomycin for 24 h in the presence or absence of CX-659S and the amount of IL-2 was measured. Data are means±SEM of three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
CX-659S inhibits production of GM-CSF from KC. Epidermal KC were prepared and cultured for 48 h with CX-659S (0–500 μM) in the presence or absence of IL-1β (100 ng per ml). The amounts of GM-CSF (A), TNF-α (B), and IL-1α (C) in each supernatant were measured. Data are means±SEM of three independent experiments. *p<0.05.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
CX-659S inhibits the phosphorylation of Erk1/2 and MEK1/2. KC were pretreated with CX-659S (500 μM) for 90 min and then stimulated with IL-1β (100 ng per ml) for the indicated periods of time. Cellular lysates were subjected to western blot analysis for Erk1/2, MEK1/2, p38 MAPK, and IκBα. Data are representative of three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
The specific MEK1/2 inhibitor U0126 mimics the effect of CX-659S. (A) Suspensions of epidermal cells were cultured with U0126 (0–50 μM) in the presence or absence of GM-CSF (20 ng per ml) for 72 h, and the expression of CD86 on Ia-positive LC was analyzed. Data are representative of three independent experiments. (B) KC were cultured with U0126 (0–50 μM) in the presence or absence of IL-1β (100 ng per ml) for 48 h, and the amount of GM-CSF in each supernatant was measured. Data are means±SEM of three independent experiments. (C) Sensitized mice were challenged by 1% PC solution in acetone to the left ear. U0126 (0–100 μg per ear) or CX-659S (0–1000 μg per ear) was painted 1 h before the challenge. The ear thickness was measured before and 24 h after the challenge, and the difference in thickness was calculated. Data are means±SEM of five mice. *p<0.05.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions