Tag reagent

Cells were loaded with 200 µM OA for 20 h and harvested. Cells were scraped from dishes, washed with PBS, and lysed in lipid extraction solution (hexane/isopropanol 3:2, vol/vol). After centrifugation at 1,000 g for 5 min, the upper organic phase was collected and dried with nitrogen gas. Lipids were resuspended in chloroform containing 1% Triton X-100 and then dried with nitrogen gas. Lipids were finally resuspended in water. TAG level was measured using TAG reagent (Sigma-Aldrich) and free glycerol reagent (Sigma-Aldrich).

Five wandering third instar larvae per genotype were collected and washed once in PBS, ethanol 70%, and water to remove food, surface bacteria, and other contaminants. Larvae were transferred to an Eppendorf tube containing 200 µl of PBS-T (PBS+0.05% Triton X-100 supplemented with protease inhibitors, Roche) and homogenized three times with a pellet pestle for 10–15 s on ice. Homogenates were subsequently sonicated. 10 µl of homogenized sample was collected for protein determination, and the rest of the homogenate was incubated at 70°C for 10 min. 20 µl of the sample and also standards were added to tubes containing 20 µl PBS-T + PI (baseline) or 20 µl TAG reagent (Sigma T2449) and incubated for 30 min at 37°C. Samples were centrifuged at maximum speed for 3 min, and 7.5 µl of each sample supernatant was transferred to clear-bottom 96-well plate containing 22.5 µl of PBS-T. 100 µl of free glycerol reagent (Sigma F6428) was added, and the mix was incubated at 37°C for 5 min. Absorbance was measured in a spectrophotometer at 560 nm. TAG concentration for each sample was determined by subtracting the absorbance for the free glycerol in the untreated samples (baseline) from the total glycerol concentration in samples that were incubated in triglyceride reagent. As a final step, samples were normalized to the corresponding protein concentrations.