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. 2016 Sep 2;6:32750. doi: 10.1038/srep32750

Figure 5. Effect of the enforced expression of BCL11B on adipocyte differentiation and the activity of adipogenic transcription factors.

Figure 5

(a–c) 3T3-L1 cells were infected with a retrovirus that expressed BCL11B-3 × FLAG or was empty (Mock). The cells were then differentiated into adipocytes, as described under “Methods.” (a) BCL11B protein content of whole cell extracts at the indicated times after treatment for adipocyte differentiation, assessed using SDS−PAGE and immunoblotting (IB) with anti-BCL11B, anti-FLAG, or anti-β-actin antibodies. Shown here are representative results of at least three replicate experiments. (b,c) Relative mRNA levels of BCL11B (b) and the adipocyte marker genes PPARγ2, perilipin, C/EBPα, and C/EBPβ at the indicated times after treatment for adipocyte differentiation, assessed using real-time PCR. All data are expressed as means ± S.E. (n = 3). **P < 0.01. (d,e) Relative luciferase activity of 3T3-L1 cells transfected with 160 ng of the indicated reporter constructs; 0, 40, or 160 ng of pBCL11B-3 × FLAG (represented as −,+ , and + +, respectively); 160 ng of pCMV-β-gal; and either 160 ng of pPPARγ and pRXRα (d) or 160 ng of the indicated C/EBPs expressing plasmid (e). The cells were either cultured with medium A for 24 h and then treated with or without 10 μM troglitazone for 24 h (d) or cultured with medium A for 48 h. Luciferase assays were performed as described under “Methods.” All data are expressed as means ± S.E. (n = 3). Bars with different letters are significantly different (P < 0.05). (f) HEK293 cells were transfected with expression plasmids for Myc-C/EBPβ and BCL11B-3 × FLAG, and the whole cell extract (WCE) was subjected to immunoprecipitation (IP) with anti-FLAG antibodies. Aliquots of whole cell extracts and pellets of immunoprecipitates were subjected to SDS-PAGE and immunoblotting (IB) with anti-c-Myc and anti-FLAG antibodies. (g) 3T3-L1 cells were differentiated into adipocytes for 24 h, and then the cells were lysed and immunoprecipitated with anti-BCL11B antibodies and immunoblotted with antibodies with anti-BCL11B and anti-C/EBP antibodies.