Bodipy 493 503

Mice were sacrificed with pentobarbital, and the heart with attached aortic roots was obtained immediately, fixed in 4% paraformaldehyde overnight, embedded in optimum cutting temperature (OCT), and then cut into serial 6-um cross sections for immunofluorescence. Lipid accumulation was stained with Bodipy 493/503 (7 μg/mL, 790389, Sigma, USA). Rabbit anti-LC3B antibody (ab48394, Abcam, UK) was used to analyze LC3B staining.

Hepatocytes were treated as described above and then fixed with 10% formalin. Lipid droplet deposition in cells was detected with BODIPY 493/503 (790389, Sigma), and then observed using a fluorescence microscope (IX51, Olympus, Japan).

Neonatal mouse cardiomyocytes were washed with 1 ml of PBS to remove the medium. The cells were incubated with 200 nM MitoTracker (Sigma) stain for 20 min in the dark at room temperature. The cells were washed with 1 ml of PBS three times. The cells were fixed with 4% paraformaldehyde for 20 min. Then, the cells were incubated with 2 μM BODIPY 493/503 (Sigma) staining solution in the dark for 20 min at 37 °C. The cells were washed using 1 ml of PBS to remove the staining solution. Finally, the nuclei were stained with Hoechst 33258 (Sigma) for 5 min. The cells were observed and photographed by fluorescence microscopy (Olympus IX83). Quantification analysis of lipid contents and mitochondria was performed using Image-Pro Plus 6.0 (Media Cybernetic Inc., USA) image analysis software.

Adipogenic differentiation was induced using adipogenic induction medium (AIM) based on DMEM/F12 medium supplemented with 2.5%FCS, 1 μM troglitazone, 500 μM 3-isobutyl-1-methyl-xanthine (IBMX), 250 nM T3, 100nM dexamethasone, and 1 μM insulin. After 7 days, AIM was exchanged for adipocyte differentiation medium (ADM) containing 2.5% FCS, 1 μM insulin, and 1 μM troglitazone, and cells were cultured until day 14. All reagents and dyes were obtained from Sigma Aldrich, Germany. For IL1B-inhibition experiments, AIM and ADM were supplemented with 100 ng/mL human recombinant interleukin 1 receptor antagonist (IL1RA, Peprotech, Austria). For microscopy assessment of lipid content, cells were stained with BODIPY-493/503 (1 μg/mL, Sigma Aldrich, Austria) and Hoechst 33342 (1 μg/mL, Sigma Aldrich, Austria) dilution in PBS for 15 min, then washed with PBS, and imaged using Celena S Digital Imaging System. Cell numbers of day 14 differentiated adipocytes were determined by counting Hoechst 33342-positive nuclei per visual field using ImageJ version 1.49m. Three random positions per well were analyzed. For flow cytometry analysis, the same cells were trypsinized, and acquisition performed using a BD-FACS Calibur flow cytometer. Analysis was completed using FlowJo Software version 10.1r1.

To visualize lipophagy induced by quercetin, AML12 hepatocytes were stained with BODIPY 493/503 (Sigma-Aldrich, St. Louis, MO, USA) for lipid droplets and LysoTracker Red DND-99 (Molecular Probes, Eugene, OR, USA) for lysosomes. Briefly, living cells were exposed to LysoTracker Red for 2 h before fixation with 4% paraformaldehyde for 15 min, followed by staining with BODIPY 493/503 for 30 min. Fluorescent images were obtained with a Leica DM5000B microscope (Wetzlar, Germany). The whole images of each well (n = 7) were analyzed by using the ImageJ plugins Colocalization_Test to evaluate colocalization of BODIPY and Lysotracker in hepatocytes (https://imagej.nih.gov/). Evaluation of colocalization was expressed by Rtotal (Pearson's correlation coefficient for the whole image, which varies between −1 and 1) divide by total BODIPY intensity.

Capsaicin (CAP), AICAR, dorsomorphin (also named compound C), troglitazone and GW9662 were obtained from Tocris (Ellisville, MO, USA). BODIPY (493/503) and STO-609 (a CaMKKβ inhibitor) were from Sigma (St. Louis, MO, USA). Anti-pAMPKα1-Thr172, anti-AMPK, anti-pACC-Ser79, anti-ACC, anti-pSREBP-1c-Ser372, anti-pAKT-Ser473, anti-AKT, anti-pmTOR-Ser2248, anti-mTOR, anti-pS6-Thr389, anti-S6, anti-p62 from Cell Signaling Technology (Danvers, MA, USA), anti-SREBP, anti-CD36, anti-PPARα from Abcam (Cambridge, UK), anti-PPARγ from Santa Cruz Biotechnology (Dallas, TX, USA), anti-PGC-1α, anti-LC3B from Novus Biologicals (Abingdon, UK) and anti-β-tubulin from Sigma (St. Louis, MO, USA).