Figure 1 Effects of jatropholone B on cell viabilityMel-Ab cells (2 × 105/well) were seeded in 24-well plates. After 24 h, cells were incubated with 1-50 μM jatropholone B for 24 h. Cell viability was measured using a crystal violet assay. Each treatment was performed in triplicate and values are represented as the mean ± standard deviation, aP < 0.01.

Figure 2 Effects of jatropholone B on melanin content in Mel-Ab cells Cells (2 × 105/well) were cultured in 6-well plates and treated with 1-50 μM jatropholone B for 4 days. A: Photographs were taken using a digital video camera. A1: Control; A2: 1 μM; A3: 5 μM; A4: 10 μM; A5: 20 μM; A6: 50 μM. B: Melanin content was measured as described in “Materials and Methods.” Each treatment was performed in triplicate, and values are represented as the mean ± standard deviation. aP < 0.01.

Figure 3 Effects of jatropholone B on tyrosinase activity Cells (2 × 105/well) were cultured in 6-well plates and treated with 1-50 μM jatropholone B for 4 d. A: tyrosinase activity was measured as described in “Materials and Methods.” B: to test the direct effect of jatropholone B on tyrosinase in a cell-free system, as described in “Materials and Methods.” Each treatment was performed in triplicate, and values are represented as the mean ± standard deviation. aP < 0.01.

Figure 4 Effects of jatropholone B on signaling pathways in Mel-Ab cells Cells (2 × 105/well) were cultured in 6-well plates. A: after serum starvation for 24 h, cells were incubated with 20 μM jatropholone B for 0-360 min. A1, A4: Western blot analysis was performed with antibodies against phospho-ERK, phospho-Akt, Akt, phospho-GSK3β, and β-catenin. A2: relative band intensity (p-ERK/total ERK); A3: relative band intensity (p-Akt/total Akt); A5: Relative band intensity (p-GSK3β/total GSK3β); A6: Relative band intensity (β-catenin/GAPDH). B: After serum starvation for 24 h, cells were incubated with 20 μM of jatropholone B for 0-72 h. B1: Western blot analysis was performed with antibodies against phospho-GSK3β and β-catenin. ERK, Akt, GSK3β and GAPDH antibodies were used as loading controls. B2: Relative band intensity (p-GSK3β/total GSK3β); B3: Relative band intensity (β-catenin/GAPDH). The band intensity relative to the control was determined by densitometric analysis. ERK: extracellular signal-regulated kinase; GSK3β: glycogen synthase kinase 3β; Values are expressed as mean ± standard deviation (n = 3). aP < 0.05 compared to untreated control.

Figure 5 Effects of jatropholone B and PD98059 on melanin synthesis. Mel-Ab cells were cultured (2 × 105/well) in 60-mm dishes, pretreated with 20 μM PD98059 for 1 h, and cultured with jatropholone B (20 μM) for 4 d. A: Photographs were taken using a digital video camera. A1: Control; A2: Jatropholone ; A3: A4: B: Melanin content was measured as described in “Materials and Methods.” Each treatment was performed in triplicate, and values are represented as the mean ± standard deviation. aP < 0.01.

Figure 6 Effects of jatropholone B and PD98059 on MITF and tyrosinase protein level A1, A2, A3: Mel-Ab cells were cultured (2 × 105/well) in 60-mm dishes and treated with 20 μM of jatropholone B for 24, 48, and 72 h. Western blot analysis was performed with antibodies against MITF and tyrosinase. B1, B2, B3: Mel-Ab cells were pretreated with PD98059 (20 μM) for 1 h and incubated with jatropholone B (20 μM) for 4 d. MITF and tyrosinase levels were measured by Western blot analysis. C1, C2, C3: Mel-Ab cells were pretreated with PD98059 (20 μM) for 1 h and incubated with jatropholone B (20 μM) for 30 min. Western blot analysis was performed using antibodies against phospho-ERK. ERK and GAPDH antibodies were used as a loading control. The band intensity relative to the control was determined by densitometric analysis. ERK: extracellular signal-regulated kinase; GSK3β: glycogen synthase kinase 3β; MITF: microphthalmia-associated transcription factor. Values are expressed as mean ± standard deviation (n = 3). aP < 0.05, compared to untreated control.