Figures & data
Fig. 1. Chemical structure of N,N,N-trimethyl phytosphingosine-iodide (TMP).
Fig. 2. Effects of L-TMP on the proliferation of NHMs.
Notes: (A) Morphologic alterations in NHMs exposed to L-TMP. (B) WST-1 cell viability assay. NHMs were treated with 0.1–10 μg/mL L-TMP for 48 h, and absorbance was measured at 450 nm using a microplate reader. *p < 0.05 vs. control (con).
Fig. 3. Effects of L-TMP on melanogenesis.
Notes: (A) NHM suspensions were photographed after 120 h of treatment without or with L-TMP. Arbutin (100 μg/mL) was used as a positive control. (B) Melanin content was measured in NHMs that were treated with L-TMP for 120 h. Data represent the mean ± SD of triplicate assays and are expressed as a percentage of control. Arbutin (100 μg/mL) was used as a positive control. *p < 0.05 in comparison with the untreated control.
Fig. 4. Effects of L-TMP on tyrosinase activity. L-TMP suppressed tyrosinase activity in NHMs.
Notes: (A) NHMs were incubated with 0–1 μg/mL L-TMP and 100 nM α-MSH for 120 h. Cellular tyrosinase activity was measured as described in the Materials and methods section. Arbutin (100 μg/mL) was used as the positive control. (B) Tyrosinase activity in a cell-free system NHMs, as described in the Materials and methods section. Each measurement was made in triplicate. Data are shown as the means ± SD. *p < 0.05 in comparison with untreated controls.
Fig. 5. Effects of L-TMP on melanogenic protein expression and the ERK, p38 and PKC pathways in NHMs.
Notes: (A) NHMs were treated with 1 μg/ml L-TMP for 24–120 h. Cell lysates were assessed using Western blot analysis using antibodies against MITF and tyrosinase. The β-actin antibody was used as a control for equal protein loading. (B) NHMs were serum-starved for 24 h and stimulated with 1 μg/mL L-TMP at the indicated times. Cell lysates were then assessed using Western blot analysis using antibodies against phospho-specific ERK and phospho-specific p38. The β-actin antibody was used as a control for equal protein loading. (C) NHMs were treated with 0.1 or 1 μg/mL L-TMP for 48 h. The PKC-β II level was determined using Western blot analysis, and β-actin was used as the loading control. (D) PKC activity was measured in NHMs using various concentrations of L-TMP (10, 25, 50, and 100 μg/mL), as described in the Materials and methods section.
Fig. 6. Effects of ERK inhibitor (PD98059) on the L-TMP-induced downregulation of melanogenesis.
Notes: (A) Cells were pretreated with 10 μM PD98059 for 30 min and then cultured with 1 μg/mL L-TMP for 120 h. The level of phospho-specific ERK was determined using Western blot analysis. Equal protein loading was confirmed by incubation with the β-actin antibody. (B) Cells were cultured with 1 μg/mL L-TMP for 120 h in the absence or presence of 10 μM PD98059. Melanin content was also measured. Values represent the means of three independent experiments ± SD. *p < 0.05 in comparison with the untreated control. (C) Tyrosinase activity was also measured. Values represent the means of three independent experiments ± SD. *p < 0.05 in comparison with untreated controls. (D) Cell lysates were subjected to Western blot analysis using antibodies against MITF and tyrosinase. The β-actin antibody was used as a control for equal protein loading.
