Figure 1.
Effects of l-arginine on fibroblast proliferation.
(A) This photograph shows human dermal fibroblast at 24 h after treatment with or without 6 mM l-arginine. DMEM with 10%FBS was used as positive control. (B) Fibroblast proliferation was determined by MTS assay after treatment with the indicated concentrations of l-arginine (0–7 mM). Results are expressed as the mean ± SEM of 3 independent experiments. *p<0.05, **p<0.01, as compared with the control group (Student’s t test). (C) The number of trypan blue negative fibroblast at 0, 6, 12 and 24 h after treated with 0 or 6 mM l-arginine. **p<0.01, as compared with the control group (Student’s t test).
Figure 2.
Effects of l-arginine deprivation and stimulation on apoptosis in fibroblasts.
(A) Apoptosis was measured using the TUNEL assay in l-arginine-deprived and -treated fibroblasts. (B) Quantification of the data from (A). **p<0.01, as compared with the control group (Student’s t test). (C) The number of cell death using the 0.1% trypan blue staining in l-arginine-deprived and -treated fibroblasts. **p<0.01, as compared with the control group (Student’s t test).
Figure 3.
Effects of l-arginine stimulation on the activities of ERK, Akt, PKA and CREB.
(A, B, C, D) ERK, Akt, PKA and CREB activities were analyzed by immunoblotting at 5, 15, and 30 min after stimulation with 6 mM l-arginine. Densitometry measurements for p-ERK, p-Akt, p-PKA and p-CREB were normalized to the amount of total ERK, Akt, PKA and CREB, respectively. (E) Fibroblasts were treated with 10 μM U0126, and then cells were stimulated with 6 mM l-arginine. The activities of CREB were analyzed by immunoblotting. Results are presented as the fold change compared with untreated cells. Data are expressed as the mean ± SEM of 3 independent experiments (*p<0.05, **p<0.01, Tukey’s post hoc test).
Figure 4.
Effects of knockdown of GPRC6A on the activities of ERK, Akt, PKA and CREB in l-arginine-treated fibroblasts.
(A, B, C, D) Fibroblasts were treated with si RNA GPRC6A and then cells were stimulated with 6 mM l-arginine. The activities of ERK, Akt, PKA and CREB were analyzed by immunoblotting. Densitometry measurements for p-ERK, p-Akt, p-PKA and p-CREB were normalized to the amount of total ERK, Akt, PKA, and CREB, respectively. Results are presented as the fold change compared with the control group. Data are expressed as the mean ± SEM of 3 independent experiments (**p<0.01, Tukey’s post hoc test).
Figure 5.
Effects of inhibition of ERK, Akt, PKA, CREB and GPRC6A on the proliferation of l-arginine-treated fibroblasts.
Fibroblast proliferation was measured by MTS assay after pretreatment with 0 or 6l-arginine and subsequent treatment with 10 μM U0126, LY294002, H-89, siRNA CREB and siRNA GPRC6A. Results are expressed as the mean ± SEM of 3 independent experiments.