Supplementary Figure 1: Adipocyte specific inducible knockout of C/EBPα in maturing and mature adipocytes.

a. Inducible, mature adipocytes specific knockout of C/EBPα. In the Adn-C/EBPα^flox/flox mice, C/EBPα is expressed normally in every cell. On doxycycline treatment, rtTA will activate the TRE promoter to induce Cre expression. Cre protein will subsequently cut the floxed region in C/EBPα and eliminate the expression of C/EBPα in all existing mature adipocytes (Adn-C/EBPα^−/−). Mice lacking TRE–Cre or Adn-rtTA were used as controls. b. Tissue profile of C/EBPα mRNA expression by qPCR analysis after 7 days of doxycycline chow diet feeding. n = 4 male mice (control group), n = 2 male mice (Adn-C/EBPα^−/− group). This experiment is representative of two independent experiments. c. qPCR analysis of C/EBPα mRNA levels in the adipocyte fraction and SVF fraction of sWAT and eWAT (left) and of C/EBPβ mRNA level in the adipocyte fraction of sWAT and eWAT (right) after 7 days of doxycycline chow diet feeding. n = 3 male mice per group. **, P < 0.001 for sWAT adipocyte; *, P = 0.04 for eWAT adipocyte. This experiment is representative of two independent experiments. d. Western-blots of C/EBPα andβ-actin levels in protein extracts of sWAT, eWAT and liver from Adn-C/EBPα^−/− mice and their control littermates after 4 days of doxycycline chow diet feeding. These images are representative of two independent western-blots experiments. e. qPCR analysis of C/EBPα, PPARγ, adiponectin and rtTA mRNA levels during day 0–day 6 of SVF differentiation. SVF is extracted from sWAT of Adn-C/EBPα^flox/flox mice. These images are from a single experiments. f. Oil Red O staining of SVF differentiation (extracted from sWAT of Adn-C/EBPα^flox/flox mice) on days 0–6, as indicated. These images are representative of two independent western-blots experiments. g. CHIP assay in adipocytes differentiated from SVF of Adn-C/EBPα^flox/flox mice. Starting from the 8th way of differentiation, doxycycline (10 μg ml⁻¹) was added to the cell growth medium for 4 days to generate C/EBPα free adipocytes and medium was switched to doxycycline free for another two days of before sample collection. CHIP was performed with C/EBPα (left), C/EBPβ (middle) and PPARγ (right) antibodies and the enrichments of Cd36 and C/ebpβ promoter in the immunoprecipitated DNA were tested by qPCR analysis, using 18s as a quality control. n = 310 cm dishes per group for C/EBPα CHIP; n = 310 cm dishes per group for PPARγ CHIP, Cd36; n = 210 cm dishes (control group), n = 310 cm dishes (Adn-C/EBPα^−/− group) for PPARγ CHIP, C/ebpβ. n = 210 cm dishes per group for C/EBPβ CHIP. *, P = 0.02 compared to control group. All data represent the mean or mean ± s.e.m. Student’s t-test. This data is from a single experiment.