Journal: The Journal of Biological Chemistry
Article Title: Cell-state-dependent regulation of PPARγ signaling by the transcription factor ZBTB9 in adipocytes
doi: 10.1016/j.jbc.2024.107985
Figure Lengend Snippet: ZBTB9 is a positive regulator of PPARγ signaling in mature adipocytes. A , Co-IP using anti-RXRα antibody of exogenously expressed proteins as indicated in differentiated 3T3-L1 cells. B , PPRE luciferase reporter activity from 293T cells transfected with an empty vector, Zbtb9 cDNA, Pparg cDNA as indicated. Cells were treated with one μΜ PPARγ antagonist T0070907 (PPARγi) or DMSO as a control (Two-way ANOVA, a Treatment∗overexpression p < 0.0001, b Treatment p < 0.0001, c Overexpression p < 0.0001, followed by Sídak's multiple comparisons test). C , Pparg gene expression measured by qRT-PCR and ( D ) PPARγ protein level in 293T cells transfected with plasmids as indicated (Two-way ANOVA, a Treatment∗overexpression p not significant, b Treatment p not significant, c Overexpression p < 0.0001, followed by Sídak's multiple comparisons test). Cell lysates were subjected to SDS-PAGE and western blotting for PPARγ. Vinculin represents a loading control. E , PPARγ protein levels from panel D were quantified by Image J (Two-way ANOVA, a Treatment∗overexpression p not significant, b Treatment p not significant, c Overexpression p < 0.0001, followed by Sídak's multiple comparisons test). F , PPRE luciferase reporter activity from differentiated 3T3-L1 adipocytes transfected with an empty vector, Zbtb9 cDNA, Pparg cDNA as indicated. G , Zbtb9 was knocked down in differentiated 3T3-L1 adipocytes with 2 independent shRNAs targeting Zbtb9 (shRNA #1, shRNA #2) and compared to the control shRNA (shCtrl). Gene expression was measured by qRT-PCR. H , Pparg gene expression and ( I ) protein level (PPARγ2 and PPARγ1) in Zbtb9 -KD 3T3-L1 mature adipocytes and control cells. Vinculin represents a loading control. J , PPARγ2 and PPARγ1 protein levels from panel I were quantified by Image J. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns: not significant.
Article Snippet: PPARγ antagonist T0070907 was added to the cell culture 4h after transfection (1 μΜ, #10026, Cayman Chemical, Ann Arbor, MI).
Techniques: Co-Immunoprecipitation Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Control, Over Expression, Gene Expression, Quantitative RT-PCR, SDS Page, Western Blot, shRNA
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