Figure 1.
NIH/3T3 cells formed insulin responsive adipocytes after prolonged induction.
(A) The whole cell culture dish view of the oil red O stain of NIH/3T3 cells induced with (R7) or without rosiglitazone (R-7) for 7 days and regular 3T3-L1 (3T3-L1) adipocytes. (B) Oil red O stained images of NIH/3T3 cells induced without rosiglitazone for 7 (R-7) or 14 days (R-14) and with rosiglitazone for 7 days (R7). (C) The glucose uptake rate in response to insulin with the standard errors (n = 3) of measurement of cells in panel (B).
Figure 2.
NIH/3T3 adipocytes did not express C/EBPα.
The relative mRNA levels and the standard errors (n = 3) of measurement of 3 adipocyte marker genes, (A) Fabp4 (Ap2), (B) Cebpa (C/EBPα) and (C) Pparg (PPARγ) were determined daily by qPCR during adipogenesis in 3T3-L1 and NIH/3T3 (R7) cells. Because the required induction times were different in these 2 cell lines, day 0 on the x-axis indicated the day the induction medium was removed and the cells were incubated in maturation medium. (D) Western blot of labeled genes in 3T3-L1 and NIH/3T3 fibroblasts and adipocytes. HSP70 was used as the internal control. Lanes 1: 3T3-L1fibroblasts, 2: NIH/3T3 fibroblasts, Lanes 3: 3T3-L1adipocytes, 4: NIH/3T3 adipocytes.
Figure 3.
C/EBPα was not required for adipogenesis in NIH/3T3 cells.
(A) The whole cell culture dish view in panel (A) and micrographs in panel (B) of the oil red O staining of 3T3-L1 (L1) and NIH/3T3 (NIH) cells induced after the cells were infected with lentiviruses expressing shRNA targeting luciferase (Luc), C/EBPα (C1 and C5) or PPARγ (P3 and P5). (C) The 2-deoxyglucose uptake and the standard errors (n = 3) of measurement of NIH/3T3 adipocytes infected with lentiviruses expressing shRNA targeting luciferase (-Luc) or C/EBPα (-C/EBP) before (f-) or after (a-) differentiation of NIH/3T3 cells.
Figure 4.
Temporal expression profiles of the C/EBPs during differentiation of 3T3-L1 and NIH/3T3 cells.
The mRNA levels and the standard errors (n = 3) of measurement of (A) C/EBPα, (B) C/EBPβ, and (C) C/EBPδ were determined by qPCR and normalized by Gapdh signal on day 0 (D0), 2 (D2) and 4(D4) in differentiation medium and day 8 in DMEM +10% FBS medium.
Figure 5.
Comparison of PI3K/AKT signaling in NIH/3T3 and 3T3-L1 adipocytes.
(A) Western blotting of phosphorylated-IRS-1(pIRS-1) and phosphorylated-insulin receptor (pIR), insulin receptor (IR), IRS-1, PDK1, PI3K,phospho-AKT (pAKT), total AKT (AKT), and β-actin in NIH/3T3 and 3T3-L1 adipocytes treated with (for 15 minutes) or without insulin. β-actin was the internal loading control. Labels were: L-Ins : 3T3-L1 -insulin, L+Ins: 3T3-L1 +insulin, N-Ins: NIH/3T3 -insulin, N+Ins: NIH/3T3 +insulin. pIRS-1 and pIR were blotted with 4G10 antibody, the rest of the proteins were probed with the antibody against the labeled protein. The blots were representative of 3 independent experiments. (B) The average band intensity normalized to beta-actin and the corresponding standard errors (error bars) were calculated after densitometry determination of each band in 3 independent blots as described in (A). None of the bands showed statistical significant difference in intensity.
Figure 6.
NIH/3T3 adipocytes induced without rosiglitazone expressed more Pparg (PPARγ) and Agt (Angiotensiongen).
The mRNA levels and the standard errors (n = 3) of measurement of selected genes involved in blood pressure regulation, glucose transport and lipid metabolism in NIH/3T3 adipocytes induced in the presence (+Rosi) or absence (-Rosi) of rosiglitazone were compared with 3T3-L1 adipocytes. The gene symbols used were those approved by the HUGO gene nomenclature committee (HANG) for the mouse. Asterisks on top of the graph bars indicated the difference between (+Rosi) and (-Rosi) cells was significant after Bonferroni correction for multiple comparison (p <0.0045).
Figure 7.
The relative mRNA levels and the standard errors (n = 3) of measurement of Cebpa (C/EBPα), Pparg (PPARγ), Fabp4 (Ap2) genes were determined by qPCR and in adipose tissues taken from 8 weeks old male C57BL/6 mice.
G: epididymal fat, M: mesenteric fat, I: interscapular fat, S: posterior thigh fat.