Fig. 4. Differentiation from pre-adipocyte to adipocyte is regulated by a single module consisting of multiple positive-feedback loops connecting back to PPARG.

(A) Test for feedback connectivity by directly activating PPARG. The PPARG agonist rosiglitazone was titrated into the medium of undifferentiated OP9 cells, and the protein abundance of the candidate feedback loop mediators was measured by SRM-MS 48 hours later, a time point at which PPARG is maximally expressed (7). Curves show the best fits to the data using an optimized Hill coefficient (black) versus a Hill coefficient of 1 (red). (B) Schematic of the seven identified feedback loops. (C) Contributions of the identified feedback loops to switching pre-adipocytes (low PPARG peak) to adipocytes (high PPARG peak) and to preventing de-differentiation. To measure contributions to differentiation, we transfected OP9 cells with siRNA 24 hours before the start of the experiment to remove the specified feedback loop components. The cells were then stimulated with 1 μM rosiglitazone for 24 hours and fixed (left column). To measure contributions to de-differentiation, OP9 cells were first differentiated into adipocytes by adding 1 μM rosiglitazone to the culture media for 48 hours, then transfected with the specified siRNA and fixed 24 hours later (right column). PPARG abundance was quantified by immunocytochemistry staining with anti-PPARG and then imaged.