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. 2019 Feb 10;41:134–145. doi: 10.1016/j.ebiom.2019.02.015

Fig. 3.

Fig. 3

Src suppression of PPARγ transcriptional activity in a kinase activity-independent manner. PPARγ transcriptional activity was assessed using luciferase assay in which both PPARγ and TK-PPRE3x-luc constructs were co-transfected into HEK293 cells with various forms of kinase constructs including Src, EGFR, and c-Abl as indicated in each figure. (a) Src repression of PPARγ transcriptional activity in a dose-dependent manner of wtSrc-GFP construct. Note that the amount of PPARγ is 0 or 600 ng, and wtSrc-GFP is increased as 0, 6, 60, 300, or 600 ng, respectively. (b) Src specific-suppression of PPARγ transcriptional activity. Luciferase assay was carried out for PPARγ transcriptional activity in the presence of several oncogenic tyrosine kinases including cAbl (Δ1-81), EGFRvIII and wtSrc-GFP. (c) Rescue of PPARγ activity by Src-specific inhibitors. Included are Src specific inhibitors, SU6656 and PP2, targeting kinase activity. (d-e) Src inhibition of PPARγ transcriptional activity independent of PPARγY78 phosphorylation (d) and also independent of Src kinase activity (e). Luciferase assay was performed in various conditions including PPARγY78F construct in the presence of wtSrc-GFP (d) or wt-PPARγ construct in the presence of different mutant forms of Src (e). Note that included are kinase-dead Src K295M-GFP, constitutive active Src Y527F-GFP constructs, or SrcW118A and SrcR175A Src mutants for SH2 or SH3 domain, respectively. (f) Both wildtype Src and kinase-dead Src suppress PPARγ activity. Using luciferase assay, PPARγ transactivation was assayed with wildtype or kinase-dead Src construct upon treatment of two specific inhibitors. (g) Src suppression of PPARγ transactivation is independent of Stat3 activity. PPARγ transactivation was assayed with Stat3 co-transfection in the presence or absence of 2 μM Stattic treatment targeting Stat3 for assessing PPARγ transcriptional activity. Note that all transfections in HEK293 cells were confirmed using immunoblot assay in each figure. RLU indicates relative light unit. Similar results were observed in at least two independent experiments.