Fatty acid free bsa

Tissues were minced into small pieces and incubated with DMEM-high glucose (FUJIFILM Wako Pure Chemicals Corporation, Osaka, Japan) containing 1% fatty acid-free BSA (FUJIFILM Wako Pure Chemicals Corporation) and 2 mg/ml collagenase (FUJIFILM M Wako Pure Chemicals Corporation) at 37°C for 1 h while shaking at 90 cycles/min. The suspension was filtered through a 200-μm nylon filter and centrifuged at room temperature at 120 g for 5 min. The floating cells were collected as the mature adipocyte fraction. The pellet was re-suspended in a hemolytic buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.4), and passed through a 25-μm nylon filter. The filtrate was then centrifuged at 120 g for 5 min, and the pellet obtained represented the SV cells.

Fatty acid-free BSA was obtained from Wako Pure Chemical Industries, Ltd., and oleic acid, AICAR and Compound C were purchased from Sigma.

H4IIE cells, a rat hepatoma cell line, were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) with 10% (v/v) fetal bovine serum (Hyclone, Rockford, IL, USA), penicillin (100 UI/mL), and streptomycin (100 UI/mL). All cell cultures were maintained in a 37°C incubator with 5% (v/v) CO₂. To induce cellular damage, 250 μM PA (Sigma, St. Louis, MO, USA) was added to serum-free DMEM medium after the cells grew to ~70–80% confluence. PA-BSA (bovine serum albumin) conjugate was prepared as described previously [17 (link)]. In brief, a 100 mM solution of PA in 0.1 M NaOH was incubated at 80°C, and fatty acid soaps were then complexed with 10% (w/v) BSA in phosphate buffered saline (PBS) at a 3.5 : 1 molar ratio of PA to fatty acid free BSA (Wako, Japan). The BSA was used as a vehicle control. CQ (Sigma, USA) was used to block lysosomal function, and the later degradation stage of autophagy was used to measure autophagic flux in cells.

MCF-7 and 3T3-L1 cells were cultured in serum-free media containing 0.2% (w/v) fatty acid-free BSA (FUJIFILM Wako) for 20 h. 3T3-L1 cells were treated with 1 μM ROSI (FUJIFILM Wako) as indicated. RNA isolation, cDNA synthesis, and qPCR were then performed as previously described^{63 (link)}. Briefly, RNA was isolated from the cells by using TRIzol reagent (Thermo Fisher Scientific). The quality and concentration of the isolated RNA were estimated by measuring the absorbance at 260 and 280 nm on a Nanodrop One spectrophotometer (Thermo Fisher Scientific). cDNA was then synthesized with PrimeScript RT reagent kit (TaKaRa Bio). The relative expression of gene was quantified by qPCR using a TB Green Premix Ex Taq II (TaKaRa Bio), which was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) in accordance with the manufacture’s protocols. The primers used are listed in Supplementary Table 1. An average threshold cycle (Ct) value was calculated and normalized to that of housekeeping gene GAPDH or Gapdh to obtain the ΔCt value.

Incorporation of NBD-phospholipids was analyzed by flow cytometry as described^{9 (link)}. In brief, HeLa cells were detached from dishes in PBS containing 5 mM EDTA, and then collected by centrifugation. Ba/F3 cells were collected from suspension culture by centrifugation. Cells (2 × 10⁵ cells per sample) were washed and equilibrated at 15 °C for 30 min in 100 μl of Hank’s balanced salt solution (pH 7.4) containing 1 g/l glucose (HBSS-glucose). An equal volume of 2 μM NBD-phospholipid in HBSS-glucose was added to the cell suspension and incubated at 15 °C. At each time point, 200 μl of the cell suspension was mixed with 200 μl of ice-cold HBSS-glucose containing 5% fatty acid-free BSA (Wako) to extract NBD-lipids incorporated into the exoplasmic leaflet of the plasma membrane, as well as unincorporated lipids. Next, the cells were analyzed with a FACSCalibur (BD Biosciences) to measure fluorescence of NBD-lipids incorporated and translocated into the cytoplasmic leaflet of the plasma membrane. Graphs for NBD-lipid flippase activities are expressed as averages ± SD from at least three independent experiments.