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First published online 6 January 2009
doi: 10.1242/jcs.026179

Journal of Cell Science 122, 401-413 (2009)
Published by The Company of Biologists 2009
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Dynamic complexes of A-type lamins and emerin influence adipogenic capacity of the cell via nucleocytoplasmic distribution of β-catenin

Katarzyna Tilgner*, Kamila Wojciechowicz*, Colin Jahoda, Christopher Hutchison and Ewa Markiewicz{ddagger}

The School of Biological and Biomedical Sciences, The University of Durham, South Road, Durham DH1 3LE, UK


Figure 1
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  		Fig. 1. Expression of PPAR{gamma} in skin dermal fibroblasts. (A) PCR with primers specific for β-actin and PPAR{gamma}. (B) Cells were co-stained with antibodies against lamin A/C and PPAR{gamma}. (C) PPAR{gamma} activity in control and emerin-null fibroblasts. Luciferase activity of control cells in GM was assigned a value of 100%. (D-G) Control and emerin-null fibroblasts were cultured in growth medium or preadipocyte differentiation medium. (D) mRNA levels of β-actin and PPAR{gamma} analysed by PCR. (E,F) Immunoblotting with antibodies against β-actin and PPAR{gamma}. (G) Cells were transfected with TK-PPRE-luciferase reporters. Bars: 10 µm; *P<0.05; **P<0.01.

 

Figure 2
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  		Fig. 2. Emerin-dependent changes in β-catenin activity and localisation in preadipogenic medium conditions. Control and emerin-null fibroblasts were cultured in growth medium or preadipocyte differentiation medium for 72 hours. (A) PCR with primers specific for β-actin and β-catenin. (B) β-catenin activity measured with TOP-FLASH reporters. (C,D,F) Immunoblots of whole-cell extracts with β-actin, total β-catenin and active β-catenin antibodies. (E,G) Cytoplasmic (cyt) and nuclear (nuc) fractions probed for total and active β-catenin. (H) Cells were co-stained with antibodies against active β-catenin and PPAR{gamma}. Bars: 10 µm; *P<0.05.

 

Figure 3
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  		Fig. 3. Enhanced adipogenesis in emerin-null fibroblasts occurs in the presence of activated β-catenin. Fibroblasts were cultured in growth medium or adipogenic medium for 120 hours. (A) PCR with primers specific for β-actin and β-catenin. (B,C,D) Immunoblotting with antibodies against β-actin, total β-catenin and active β-catenin. (E,F) Cells were transfected with TOP FLASH or TK-PPRE-luciferase reporters. (G) PCR with primers specific for β-actin and PPAR{gamma}. (H) Immunoblotting with antibodies against β-actin and PPAR{gamma}. (I) Fibroblasts were stained with Oil-red O. (J) Cells were grown in adipogenic medium with 10 mM LiCl (AM+LiCl) and stained with Oil-red O or probed with antibodies against β-catenin and β-actin. (K) Oil-red O was extracted with 100% isopropanol and quantified in spectrophotometer at 500 nm. *P<0.05, **P<0.01.

 

Figure 4
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  		Fig. 4. Enhanced stability of nuclear β-catenin in the absence of emerin. (A-C) 10 µM of cyclohexamide or 10 µM of MG-132 was added to the medium. Cells were harvested at intervals of 1 hour and analysed for active β-catenin by immunoblotting. (D) PCR with primers specific for β-actin and β-catenin.

 

Figure 5
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  		Fig. 5. Changes in emerin and lamins A/C expression upon downregulation of β-catenin with siRNA. (A) PCR with primers specific for β-actin and β-catenin. (B,E) Emerin and lamins A/C expression by RT-PCR against β-actin. (C,F) Immunoblotting of whole-cell extracts or nuclei with emerin and lamins A/C antibody, together with β-actin loading control. (D,G) Cells were stained with emerin or lamins A/C antibodies. Bars: 10 µm.

 

Figure 6
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  		Fig. 6. Transfection with β-catenin siRNA rescues adipogenesis in emerin-null cells. (A) PCR with primers specific for β-actin and PPAR{gamma}. (B) Immunoblotting with β-actin and PPAR{gamma} antibodies. (C) Activity of PPAR{gamma} in GM and AM upon β-catenin knockdown. (D,E) siRNA-transfected cultures were stained with Oil-red O. *P<0.05, **P<0.01, ***P<0.001.

 

Figure 7
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  		Fig. 7. Dynamic changes in lamins A/C and emerin expression in different stages of adipogenesis. Control and emerin-null fibroblasts were cultured in growth medium (GM), then preadipocyte differentiation medium (PRE) for 72 hours or adipogenic medium (AM) for 120 hours. (A) β-actin loading controls by PCR and immunoblotting. (B,C) Emerin expression by RT-PCR and immunoblotting. (D) Cells were co-stained with PPAR{gamma} and emerin antibodies. (E,F) Lamins A/C expression by RT-PCR and immunoblotting. (G) Cells were stained with lamins A/C antibodies. Bars: 10 µm.

 

Figure 8
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  		Fig. 8. Adipogenesis in committed preadipocytes is accompanied by changes in emerin and lamin A expression. 3T3-F442A preadipocytes were cultured in growth medium (GM), preadipocyte differentiation medium (PRE) for 72 hours or adipogenic medium (AM) for 120 hours or transfected with scrambled, emerin-specific or β-catenin-specific siRNA. (A,E) PCR with β-actin, β-catenin, emerin and lamins A/C-specific primers. (B,F) Immunoblotting with β-actin, β-catenin, emerin and lamins A/C antibodies. (C,G) PCR with PPAR{gamma}-specific primers. (D,H) Oil-red O staining of the cultures. (I) Oil-red O was extracted with 100% isopropanol and quantified in spectrophotometer at 500 nm.

 

Figure 9
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  		Fig. 9. Proper execution of adipogenesis requires emerin and lamina remodelling. In early stages of adipogenic activation, upregulation of emerin expression could contribute to efficient re-distribution of β-catenin from nucleus to the cytoplasm. In skin fibroblasts, this is additionally associated with increased expression of lamins A/C. These changes could facilitate the proteasomal degradation of β-catenin, activation of PPAR{gamma} and decreased expression of lamins A/C in later stages of adipogenesis. In addition to proteasomal degradation of β-catenin, downregulation of both β-catenin and emerin expression, accompanied by increased expression of lamins A/C, could also result in early onset of PPAR{gamma} activation and adipogenesis. This feedback mechanism would ensure correct response to the environmental signals influencing extent of adipogenesis.

 

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© The Company of Biologists Ltd 2009