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. 2016 May 13;5(7):491–505. doi: 10.1016/j.molmet.2016.05.004

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© 2016 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Overexpression of PPARγ2 increases the proportion of lipid laden-cells and the gene expression profile but not the morphology of differentiated Agpat2^−/−MEFs. (A) qPCR quantification of C/EBPs and PPARγ mRNA levels at early and late stages of adipogenic differentiation. Data were normalized to 36B4 mRNA levels and expressed as relative fold changes to non-differentiated Agpat2^+/+ MEFs at day 0. (B) Immunoblot analysis of whole-cell protein extracts from Agpat2^+/+ and Agpat2^−/− MEFs at different days of differentiation. 50 μg of proteins were loaded, β-actin was used as loading control. (C) Confocal immunofluorescence analysis of PPARγ in Agpat2^+/+ and Agpat2^−/− MEFs at two different stages of adipocyte differentiation. Graphs show the relative total immunofluorescence signal of nuclear PPARγ per cell. (D) Time-line showing the adipogenic differentiation protocol used on primary cultures of Agpat2^+/+ and Agpat2^−/− MEFs previously infected with recombinant adenoviruses. (E) Adipogenic differentiation expressed as percentage of BODIPY stained cell after 6 days of differentiation. (F) Total cellular triglycerides quantified by an enzymatic-colorimetric method. (G) mRNA levels of adipogenic transcription factors and adipocyte related markers were quantified by qPCR. Gene expression was normalized to 36B4 mRNA levels and presented as fold-change relative to non-differentiated Agpat2^+/+ MEFs. (H) Three dimensional digital reconstruction of fluorescence stacks of differentiated Agpat2^+/+ and Agpat2^−/− MEFs immunostained with anti-Perilipin-1 antibody (pink). Neutral lipids were stained with BODIPY (green). All bar graphs show mean ± SD of three independent experiments (N > 6). *** (p < 0.001), **(p < 0.01) and *(p < 0.05) denote significant difference compared to Agpat2^+/+ MEFs.