Fig 1.
Stellera chamaejasme L. methanol extract suppressed the induced differentiation of 3T3-L1 preadipocytes without substantial cytotoxicity.
(A) 3T3-L1 preadipocytes were cultured in differentiation medium for 6 days in the presence of Stellera chamaejasme L. (SCL) methanol extract or vehicle (control). Cells were then stained for lipid accumulation using Nile Red, while nuclei were counterstained with Hoechst 33342. Images were acquired by epifluorescence microscopy. The scale bar represents 100 μm. (B and C) Nile Red staining intensity and cell viability were quantified using ImageJ software (n = 6 wells per treatment). All results are presented as the mean ± SEM of two independent experiments. **, p < 0.01; ***, p < 0.001 versus the control group.
Fig 2.
Stellera chamaejasme L. methanol extract suppressed the expression of adipogenic and lipogenic genes in 3T3-L1 cells cultured under differentiation induction conditions.
−Cells were cultured for 6 days in differentiation medium plus SCL or vehicle. (A) The mRNA expression levels of adipogenic genes were analyzed by quantitative real-time PCR (qRT-PCR). (B) Expression levels of PPARγ and Adiponectin proteins were determined by Western blotting. (C) The mRNA expression levels of lipogenic genes were analyzed by qRT-PCR. All results are presented as the mean ± SEM of two independent experiments (n = 4 wells per treatment). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus the control group.
Fig 3.
Stellera chamaejasme L. methanol extract interferes with multiple stages of adipocyte differentiation.
(A) Schematic representation of the experiment. Cultures of 3T3-L1 cells were treated with SCL at different times during the 6-day differentiation induction period to block specific stages. The fresh medium contained SCL extract that was changed every other day during adipogenesis. (B) After the differentiation induction period, cells were stained with Nile Red to assess adipogenesis and nuclei were counterstained with Hoechst 33342. Images were acquired by epifluorescence microscopy. The scale bar represents 100 μm. (C) The reduction in lipid accumulation differed according to the SCL extract exposure period (Condition) (n = 6 wells per treatment). (D and F) Extract treatment during the induction protocol also reduced the mRNA expression levels of adipogenic (D) and lipogenic (F) genes as estimated by qRT-PCR. (E) Expression levels of PPARγ and Adiponectin proteins were determined by Western blotting. All results are presented as the mean ± SEM of two independent experiments (n = 4 wells per treatment). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus the control group (condition 1). #, p < 0.05; ##, p < 0.01; ###, p < 0.001 versus the SCL treatment group for 6 day (condition 7).
Fig 4.
Stellera chamaejasme L. methanol extract inhibited the adipogenic differentiation of primary SVCs.
Cells were cultured under differentiation induction conditions for 6 days in the absence (control) or presence of various SCL extract concentrations. (A) After the differentiation protocol, cells were stained with Nile Red to assess lipid accumulation and nuclei were counterstained with Hoechst 33342. Images were acquired under epifluorescence microscopy. The scale bar represents 100 μm. (B) The SCL extract reduced lipid accumulation in SVC-derived adipocytes as measured by Nile Red staining (n = 6 wells per treatment). (C) The number of cells was counted by Hoechst 333342 staining (n = 6 wells per treatment). (D and F) The SCL extract also reduced the mRNA expression levels of adipogenic and lipogenic genes as estimated by qRT-PCR. (E) PPARγ and Adiponectin protein expression levels were reduced by SCL extract as evidenced by Western blotting. All results are presented as the mean ± SEM of two independent experiments (n = 4 wells per treatment). **, p < 0.01; ***, p < 0.001 versus the control group.
Fig 5.
The SCL extract enhanced mitotic clonal expansion of 3T3-L1 cells during the early period of adipocyte differentiation.
(A) 3T3-L1 preadipocytes were cultured under differentiation induction conditions for 0, 24, and 48 h in the absence (control) or presence of SCL extract. The extract reduced the mRNA expression levels of adipocyte-specific transcription factors and enhanced expression of cell cycle-related genes as estimated by qRT-PCR (n = 4 wells per treatment). (B) Representative flow cytometry images showing the cell cycle phase distribution of 3T3-L1 cells cultured under differentiation induction conditions in the absence and presence of SCL extract. (C) Quantitative analysis of flow cytometry results. The extract enhanced the proportions of cells in S phase and G2/M phase. All results are presented as the mean ± SEM from two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus each control group.
Fig 6.
The SCL extract blocked adipocyte differentiation and promoted mitotic clonal expansion of preadipocytes through activation of the ERK pathway.
(A) 3T3-L1 preadipocytes were cultured in induction medium containing various concentrations of SCL for 15 min. (B) 3T3-L1 preadipocytes were cultured in induction medium containing 33-μg/mL SCL for 15 min in the absence or presence of the ERK inhibitor U0126 (10 μM). Total ERK and phosphorylated (p)-ERK protein expression levels as determined by Western blotting. (C) The ERK inhibitor U0126 (10 μM) reversed the inhibitory effect of SCL (33 μg/mL) on adipocyte marker gene expression as determined by qRT-PCR. (D and E) The ERK inhibitor U0126 (10 μM) reversed the effect of SCL extract (33 μg/mL) on mitotic clonal expansion. (D) Representative flow cytometry images showing the cell cycle phase distribution. (E) Quantitative analysis of the cell cycle phase distribution. Cotreatment with U0126 reversed the SCL extract-induced increases in S and G2/M phase cells. All results are presented as the mean ± SEM of two independent experiments. *, p<0.05; **, p < 0.01, ***, p < 0.001 versus each control group #, p < 0.01, ###, p < 0.001.