Triolein

CCA quantification of Drosophila homogenates was essentially done as described in [22] (link). If not described differently eight flies per replicate were homogenized in a 2 ml screwcap tube containing 1 ml 0.05% Tween-20 and a ceramic cylinder using a peqlab Precellys 24 instrument (10 sec at 5000 rpm). Homogenates were heat-inactivated (5 min at 70°C) and debris pelleted in a Beckmann GS6KR centrifuge (3 min at 3500 rpm). Of the supernatants 50 µl samples were transferred to a 96 well microtiter plate and homogenate (blank) absorbance was measured at 540 nm in a Biorad Benchmark Microplate Reader. Prewarmed Triglyceride solution (200 µl; Thermo Fisher Scientific #981786) was added to each homogenate sample and incubated at 37°C with mild shaking for 30–35 min. Total absorbance at 540 nm was measured and corrected by subtraction of blank and substrate absorbance prior to triglyceride equivalent content calculation using 0–40 µg of triolein (Sigma T7140) as TAG standard, which was treated like the samples.
For experiments with inactive CCA reagent shown in Fig. 1C the Triglyceride solution was heat-inactivated (5 min at 96°C) or incubated with 200 µM of the lipase inhibitor Orlistat (Sigma O4139) prior to use.
For homogenate absorbance determination prior to CCA assay (Fig. 2A), the 540 nm absorbance of 250 µl 0.05% Tween-20 was subtracted as blank value. Homogenate absorbance values were calculated per mg fly wet weight.
For experiments shown in Fig. 2B, 16 flies per replicate were homogenized in 1 ml 0,05% Tween-20. Homogenate supernatants (150 µl) were added to equal volumes of 0.05% Tween-20 containing increasing amounts of triolein and treated once more in the peqlab Precellys 24 instrument as described. Aliquots (50 µl) of the resulting homogenate samples were subjected to CCA measurement as described.
Shown are representative experiments with average values of triplicate measurements and corresponding standard deviations. Experiments were repeated at least twice.
For fly free glycerol content determination eight male flies were homogenized in 0.5 ml 0.05% Tween-20 as described above. Free glycerol content of 50 µl homogenate supernatants was determined with the Free Glycerol Reagent (Sigma F6428) using 0–50 µg triolein equivalents (Glycerol Standard Solution, Sigma G7793) as standard. Total free glycerol and glyceride content was determined by diluting 25 µl of the aforementioned homogenate with 25 µl 0.05% Tween-20 before using the Free Glycerol Reagent combined with the Triglyceride Reagent (Sigma T2449+F6428) using 0–40 µg triolein as standard. Free glycerol content and total free glycerol+glyceride content both expressed as µg triolein equivalent/mg fly wet weight were calculated as described above.
Shown are average values of triplicate measurements of three independent experiments.

Hydrogenated soybean phosphatidylcholine (HSPC) was obtained from Japan NOF Corporation (Tokyo, Japan). Cholesterol and 1,2-dipalmitoyl-sn-3-phospho glycerol (DPPG) were from Avanti Polar Lipids (AL, USA). Triolein was purchased from Shanghai Chemical Reagent Co. (Shanghai, China). l-Lysine was purchased from Sigma Aldrich Co. (USA). Ioversol injection (320 mg/mL) and ketamine (2 mL:0.1g) were from Jiangsu Hengrui Medicine Co., Ltd. (Lianyungang, China). Doxorubicin hydrochloride was from Shenzhen Wanle Pharmaceutical Co., Ltd (Shenzhen, China) and diazepam was from Xudonghaipu Medicine Co., Ltd. (Shanghai, China). All other reagents were of chemical pure or analytical grade. All the materials and reagents were used as received without any further purification.

A double-emulsion procedure (Li et al., 2019 (link)) was used to prepare MVLs containing ioversol and doxorubicin hydrochloride. Briefly, 1 mL of chloroform containing the lipids (41 mg HSPC:40.5 mg cholesterol:5 mg DPPG: 11.25 mg triolein) in 1 mL aqueous solution (the first aqueous solution) was emulsified by Scientz-IID Ultrasonic Homogenizer (Ningbo Scientz Biotechnology Co., Ltd. Ningbo, China) for 30 sec (30% power output, 25 °C) to produce a w/o emulsion. The first aqueous solution contains 1 mg of doxorubicin hydrochloride in 1 mL of 320 mg ioversol injection. This w/o emulsion (2 mL) was subsequently emulsified with 6 mL of the second aqueous solution containing 4% glucose (wt/vol) and 20 mM lysine at 2800 r/min by XHF-D mixer (Ningbo Scientz Biotechnology Co., Ltd. Ningbo, China) at 40 °C to prepare w/o/w emulsion. Then the w/o/w emulsion was diluted with 4 mL of second aqueous solution poured into 100-mL egg type flask. Chloroform was removed by flushing nitrogen over the surface of the double emulsion at 35–37 °C. The resultant MVLs were collected at 100g for 10 min, and resuspended in sterile saline solution after discarding the supernatant. BD Falcon™ cell strainers (BD Biosciences, USA) were used to isolate different size MVLs, improve the uniformity of MVLs. The ioversol and doxorubicin hydrochloride concentration in MVLs were determined by HPLC.