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First published online 14 February 2006
doi: 10.1242/jcs.02792

Journal of Cell Science 119, 889-897 (2006)
Published by The Company of Biologists 2006
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PKB{alpha} is required for adipose differentiation of mouse embryonic fibroblasts

Anne Baudry, Zhong-Zhou Yang and Brian A. Hemmings*

Friedrich Miescher Institute for Biomedical Research, Maulbeerstr. 66, CH-4058, Basel, Switzerland


Figure 1
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  		Fig. 1. Inhibition of adipogenesis in PKB{alpha}–/– MEF cells. (A) Two days after confluence, MEF cells were induced to differentiate into adipocytes with IBMX, dexamethasone and insulin for 48 hours followed by insulin alone every 2 days. Cells were stained with Oil-Red O at 8 days post-induction. (B) Expression of adipocyte-specific genes after stimulation of the adipose differentiation process in wild-type and PKB{alpha}–/– MEFs. Total RNA was extracted from cells at the times indicated and analysed by RT-PCR as described in the Materials and Methods.

 

Figure 2
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  		Fig. 2. Levels of the three PKB isoforms and the phosphorylation status of PKB in wild-type and PKB{alpha}–/– MEFs during adipose conversion. Total cellular extracts were prepared from cells before (day 0) or at 2, 5, 8 and/or 12 days after adipose induction treatment and analysed by western blotting with PKB{alpha}-, PKBß- and PKB{gamma}-specific antibodies (A) or with phospho-PKB specific antibodies (pSer473 and pThr308) (B). Blots were reprobed with anti-actin antibodies to control for equal loading.

 

Figure 3
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  		Fig. 3. Ectopic expression of PKB{alpha} by retrovirus infection restores adipogenesis in PKB{alpha}–/– MEFs. (A) PKB{alpha}–/– MEFs were infected with retrovirus expressing HA-PKB{alpha} or vector only. After puromycin selection, extracts of cells were analysed by western blotting for the expression of HA-PKB{alpha}. (B) Cells were exposed to the adipogenic cocktail and stained with Oil Red O after 15 days. (C) RT-PCR analysis of adipose markers PPAR{gamma} and aP2 in PKB{alpha}–/– MEFs infected with vector or HA-PKB{alpha}-expressing retrovirus and submitted to adipose differentiation.

 

Figure 4
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  		Fig. 4. Cluster map of genes upregulated during an early step of adipose differentiation in wild-type MEFs showing little or no upregulation in PKB{alpha}–/– MEFs. The genes identified by microarray analysis were grouped into ten clusters according to their temporal profile of expression. Values are the average fold induction after adipogenic treatment of all genes in each cluster. Cluster 10 which comprises all otherwise unclassified genes is presented in Table 1.

 

Figure 5
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  		Fig. 5. PKB{alpha} induces Klf15 during adipose conversion of MEFs. (A) Microarray profile of Klf15 in wild-type and PKB{alpha}–/– MEFs during the first 2 days after hormonal stimulation. (B) Total RNA was extracted from cells at the times indicated and analysed by RT-PCR. (C) RT-PCR analysis of Klf15 gene expression after 24 hours of adipogenic treatment of PKB{alpha}–/– MEFs infected with vector or HA-PKB{alpha} expressing retrovirus. (D) PKB{alpha}–/– MEFs were infected with retrovirus expressing (m/p)-HA-PKB{alpha} or vector only. Following puromycin selection, lysates were analysed by western blotting for the expression of a constitutively active form of PKB{alpha}. (E) Total RNA was extracted from cells 3 days postconfluence and analysed by RT-PCR as described in the Materials and Methods.

 

Figure 6
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  		Fig. 6. Inhibition of the PI 3-kinase/PKB signalling pathway reduces expression of Lcn2, Ramp3, Ren1 and Klf15 during the first stages of adipose differentiation of 3T3-L1 cells. Microarray profiles (A) and RT-PCR analysis (B) of Lcn2, Ramp3 and Ren1 genes in wild-type and PKB{alpha}–/– MEFs during the first 2 days of adipose treatment. (C,D) 3T3-L1 cells were starved for 24 hours and then treated with LY294002 or not (DMSO only) 20 minutes before and every 12 hours after addition of the adipose cocktail. Lysates and RNA from cells were collected at the times indicated. Total cellular proteins were subjected to western blot analysis with phospho-PKB-specific antibodies (pSer473) and actin as a loading control (C) and total RNA was submitted to RT-PCR analysis for Ramp3, Lcn2, Ren1 (D) or Klf15 (E).

 




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