INTRODUCTION

METHODS
Antibodies and other reagents
Cell culture and adipocyte differentiation
Transfection
Construction and adenovirus infection
Immunoblotting
Oil Red O staining
Quantitative RT-PCR
MTT assay
Statistical analysis

RESULTS
TonEBP expression was decreased during adipocyte differentiation in 3T3-L1 cells
![]() | Fig. 1Expression of TonEBP during adipocyte differentiation.3T3-L1 preadipocytes were induced to differentiate into adipocytes. (A) Total cell lysates were prepared at indicated times and protein expression was analyzed by immunoblotting. (B) TonEBP expression (A) was normalized to that of GAPDH and then presented as a relative value based on D0. (C) Expression of TonEBP mRNA at indicated times was measured by quantitative RT-PCR and presented as a relative value to D0. GAPDH mRNA expression was used as a reference. Values are mean±SEM. *p<0.05, **p<0.01 compared with D0 for each group (n≥3).
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TonEBP suppressed adipocyte differentiation
![]() | Fig. 2Suppression of adipocyte differentiation by TonEBP.3T3-L1 cells were infected with recombinant adenovirus expressing β-galactosidase or TonEBP and then induced to differentiate into adipocytes. Expression of TonEBP (A) or adipogenic proteins (D) was analyzed by immunoblotting. (B) The degree of adipocyte differentiation was visualized by staining lipid droplets with Oil Red O at D6. (C) Lipid droplets shown in (B) were extracted with isopropyl alcohol and quantified as a relative value to Adβ-gal. (E) The quantity of FABP4 shown in (D) was normalized to that of GAPDH and then presented as a relative value to D6 of Adβ-gal. Values are mean±SEM. *p<0.05, **p<0.01 compared to Adβ-gal for each group (n=3).
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![]() | Fig. 3Promotion of adipocyte differentiation by TonEBP knockdown.3T3-L1 cells were transfected with scrambled or TonEBP siRNA and then induced to differentiate into adipocytes. Expression of TonEBP (A) or adipogenic proteins (D) were analyzed by immunoblotting. (B) The degree of adipocyte differentiation was visualized by staining lipid droplets with Oil Red O at D6. (C) Lipid droplets shown in (B) were extracted with isopropyl alcohol and quantified as a relative value to scrambled siRNA. (E) The quantity of FABP4 shown in (D) was normalized to that of GAPDH and then presented as a relative value to D6 of scrambled siRNA. Values are mean±SEM. *p<0.05, **p<0.01 compared to scrambled siRNA for each group (n≥3).
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TonEBP suppressed early responses to differentiation
![]() | Fig. 4Limited anti-adipogenic property of TonEBP in the early stages of adipocyte differentiation3T3-L1 cells were treated with differentiation factors at D0 for adipogenesis. Recombinant adenoviruses were infected at indicated times as described in (A). (B) Expression of each protein was analyzed by immunoblotting. (C) The degree of adipocyte differentiation was visualized by staining lipid droplets with Oil Red O at D6. (D) The quantity of FABP4 shown in (B) was normalized to that of GAPDH and then presented as a relative value to D-3 of Adβ-gal. Values are mean±SEM. **p<0.01, ***p<0.001 compared to Adβ-gal for each group (n=6).
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![]() | Fig. 5Suppression of MCE and insulin signaling with TonEBP overexpression.3T3-L1 cells infected with recombinant adenoviruses were treated with differentiation factors (IDI) for the indicated times. (A) Cell number was determined via MTT assay and presented as a relative value to DO. (B) The protein levels of phosphorylated IRS-1 (p-IRS-1), IRS-1, phosphorylated Akt (p-Akt), and Akt in these cells were detected by immunoblotting using specific antibodies. The experiment was repeated three times with similar results.
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DISCUSSION
