Adipocyte differentiation assay and Oil-Red O staining

Mutant and wild-type MEFs were used for assays of adipocyte differentiation in culture. Monolayer cultures at about 50% confluency were first treated for 2 days with the “Induction Medium” (DMEM supplemented with 10% FCS, 8 μg/ml biotin, 4 μg/ml pantothenate, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, 10 μg/ml insulin (Sigma-Aldrich), and 10 μM troglitazone. The induced cells were than cultured in “Differentiation Medium” (DMEM supplemented with 10% FCS, 8 μg/ml biotin, 4 μg/ml pantothenate, and 10 μg/ml insulin) for up to 12 days to achieve differentiation to adipocytes. All the compounds were purchased from Sigma-Aldrich if not specified.

Bone marrow-derived primary MSCs were used for adipocyte differentiation following the standard protocol¹⁰. Briefly, the cells at 2–3 passages in culture were differentiated into adipocyte lineage by incubation with 5 μg/ml insulin, 1 μM dexamethasone, and 5 μM IBMX for 2 days. The cells were then maintained in 10% FBS with 5 μg/ml insulin for 7 days to complete differentiation.

For adipocyte differentiation from ES cells, a published protocol was followed (10). Multiple clones of Dab2 null and positive control pluripotent ES cells were prepared by culturing for two days to remove residual MEF feeder cells²⁰. The ES cells were then maintained in suspension cultures without LIF but in the presence of 1 μM retinoic acid (RA) for 7 days to form embryoid bodies. The aggregated cells were then plated on cell culture dishes to allow adhesion and spreading, and were subsequently differentiated into adipocytes according to the standard protocol using the two-step procedure. Briefly, the adherent cells derived from embryoid bodies were primed by incubation with 5 μg/ml insulin, 1 μM dexamethasone, and 5 μM IBMX for 2 days. After this induction, cells were maintained in 10% FBS with 5 μg/ml insulin for 7 days to fully differentiate into adipocytes.

To inhibit Ras/MAPK pathway during adipocyte differentiation assays, the MEK inhibitor U0126 (Sigma-Aldrich) in DMSO was added to the cultures to a final concentration of 1 μM. It appeared that the inhibitor did not show significant cytotoxicity during the 4-day incubation.

Oil-Red O staining was performed to visualize the intracellular lipid droplets. Briefly, 0.5 g of Oil-Red O was dissolved in 100 ml isopropanol overnight with agitation. After sedimentation, the Oil-Red O solution and water were mixed in a 6:4 ratio to making the working solution. The differentiated adipocytes were fixed in 4% PFA for 1 hour after washing 3 times with PBS, air dried, and incubated for 15 min with freshly filtered Oil-Red O working solution. After removing the Oil-Red O solution and washing 3 times with PBS, the cells were imaged and quantitated using bright field microscopy. The Oil-Red O dye associated with the adipocytes was extracted with 100% isopropanol and the absorbance was measured at 550 nm.