Journal: Journal of Biological Chemistry

Article Title: The transcription factor NKX1-2 promotes adipogenesis and may contribute to a balance between adipocyte and osteoblast differentiation

doi: 10.1074/jbc.ra119.007967

Figure Lengend Snippet: Figure 5. NKX1-2 inhibits COUP-TF II expression during adipocyte differentiation of ST2 cells. A, Upper panel, RT-qPCR was performed to examine endogenous COUP-TF II mRNA expression in pMSCV-EV and pMSCV-Nkx1-2 ST2 cells during adipocyte differentiation following addition of the hormone cocktail MDI+Rosi at Day 0. Data were normalized to cyclophilin and expression of COUP-TF II at Day 0 in pMSCV-EV was set to 1. Data are given as the mean ± S.D. of triplicate samples. Statistical significance versus empty vector was evaluated at each time point with the Student’s t test: **p<0.01 and ***p<0.001. The results are representative of three independent experiments. A, Lower panel, Immunoblot was performed for COUP-TF II protein from cell lysates harvested as described in the upper panel. Beta-actin served as a loading control. B, Upper panel, RT-qPCR was performed to examine endogenous COUP-TF II mRNA expression in shControl and shNkx1-2-2 3T3-L1 cells during adipocyte differentiation after addition of the hormone cocktail MDI. Data were normalized to cyclophilin and expression of COUP-TF II at Day 0 in shControl was set to 1. Data are given as the mean ± S.D. of triplicate samples. Statistical significance versus empty vector was evaluated at each time point with the Student’s t test: **p<0.01 and ***p<0.001. The results are representative of three independent experiments. B, lower panel, Immunoblot was performed for COUP-TF II protein from cell lysates harvested as described in the upper panel. Beta-actin served as a loading control. C-E, as indicated in Figure 2, shControl (here defined as shControl 1) and shNkx1-2 3T3-L1 preadipocytes were further infected with either shControl (defined as shControl 2) or shCOUP-TF II expression vectors, followed by adipocyte differentiation induced by MDI (as described in Figure 2) up to Day 8. C, Cells were stained with Oil Red O (ORO) to assess neutral lipid accumulation. All panels are the same magnification; a 100 µm scale bar is shown in the lower right panel. D, Upper panel RT-qPCR was performed to examine endogenous COUP-TF II mRNA expression; Lower panel, immunoblots were performed using the antibodies indicated. E, RT-qPCR was performed to examine adipocyte gene expression. For D and E, data were normalized to cyclophilin and expression of COUP-TF II, PPARγ, FABP4 or C/EBPα p30 in double shControls was set to 1. Data are given as the mean ± S.D. of triplicate samples. Statistical significance versus empty vector was evaluated at each time point with the Student’s t test: **p<0.01 and ***p<0.001. The results are representative of three independent experiments.

Article Snippet: Antibodies against the following proteins were obtained as indicated: PPARγ (H-100, sc7196), C/EBPα (14AA, sc-61), GAPDH (6C5, sc32233) and NKX2-1/TTF-1 (H190, sc-13040) from Santa Cruz Biotechnology; FABP4 (Cat# MAB1443) from R&D Systems, Inc.; NKX1-2 (ab105940) from Abcam; β-actin (Cat# 4967), COUP-TF II (Cat# 6434) and HA-Tag (C29F4, Cat# 3724) from Cell Signaling Technology.

Techniques: Expressing, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Control, Infection, Staining, Gene Expression