Fig. 2: ACSS2 binds PPARγ to transactivate Ucp1.

A 6–8-week-old wild type or Acss2^−/− male mice were challenged with or without cold exposure at 4 °C for 16 h. The mRNA levels of Ucp1 in BAT and ingWAT were shown ( = 3 per group). B The differentiated beige adipocytes in vitro from the stromal vascular fraction (SVF) of the ingWAT in wild type mice were transfected with 2 μg ACSS2 or control plasmids for 24 h. The mRNA and protein levels of ACSS2 and UCP1 were evaluated by qRT-PCR and western blot (n = 3 biological replicates). C The mRNA and protein levels of ACSS2 and UCP1 were then evaluated in the differentiated beige adipocytes from wild type or Asss2^−/− mice by qRT-PCR and western blot (n = 3 biological replicates). D The luciferase activities of Ucp1 promoter in the presence or absence of PPARγ and ACSS2 in HEK293T cells were examined (n = 6 biological replicates). E Representative immunoblot of endogenous immunoprecipitation (IP) in BAT and ingWAT (n = 3 biological replicates). F Representative immunoblot of exogenous IP of FLAG-tagged ACSS2 or HA-tagged PPARγ, FLAG-tagged RXRα, CEBPβ and CREB in HEK293T cells (n = 3 biological replicates). G Representative fluorescent images of HEK293T cells transfected with 2 μg plasmid encoding ACSS2 or PPARγ fused to the fluorescent protein fragments indicated in each panel. DAPI stain demonstrated nuclear locus. The intensity YFP signal indicates the amounts and localization of BiFC complex (ACSS2-PPARγ) (n = 3 biological replicates). H Representative FRET images of HEK293T cells transfected with 2 μg plasmid encoding ACSS2 or PPARγ fused to the EGFP and mCherry indicated in each panel. The images before and after acceptor-photobleaching were shown as well and the FRET efficiency in the nuclear was calculated from individual cells (n = 25). I Representative immunoblot of pull-down assay of purified recombination proteins His-ACSS2 and GST-PPARγ from BL21 (DE3) (n = 3 biological replicates). J Representative immunoblot of exogenous IP of FLAG-tagged full length ACSS2, HAN or HAC with HA tagged PPARγ in HEK293T cells (n = 3 biological replicates). K Representative immunoblot of exogenous IP of FLAG-tagged ACSS2 and HA-tagged PPARγ or its LBD in HEK293T cells (n = 3 biological replicates). L Representative immunoblot of exogenous IP of FLAG-tagged ACSS2 and HA-tagged PPARγ or its mutants in HEK293T cells (n = 3 biological replicates). M Representative BiFC images of HEK293T cells transfected with ACSS2 or its truncated mutants and PPARγ fused to the fluorescent protein fragments indicated in each panel (n = 3 biological replicates). N Representative BiFC images of HEK293T cells transfected with ACSS2 and PPARγ or its LBD fused to the fluorescent protein fragments indicated in each panel (n = 3 biological replicates). O Representative BiFC images of HEK293T cells transfected with ACSS2 and PPARγ or its mutants fused to the fluorescent protein fragments indicated in each panel (n = 3 biological replicates).