Bodipy 558 568 c12

Wild-type and gene-integrated hMSCs were cultured in StemPro adipogenesis, osteogenesis, or chondrogenesis induction media according to manufacturer protocols (ThermoFisher Scientific). Adipogenic differentiation cultures were stained for lipid droplets with BODIPY558/568-C12 or LipidTOX-Red dye (ThermoFisher Scientific) at 7–14 days of culture. Osteogenic differentiation cultures were stained with Alizarin Red (Millipore Sigma, Burlington, MA) solution for deposited calcium after 21 days of culture. Chondrocyte pellets were stained with Alcian Blue (Sigma-Aldrich) for proteoglycans and embedded in optimal temperature cutting compound (Fisher Scientific) and frozen sections were obtained for microscopy. Fluorescence and brightfield images of MSCs in culture vessels were obtained with ThermoFisher Scientific EVOS FL imaging system for GFP and fluorescent dye visualization. Light microscopy for colored dye stained osteogenesis cultures and frozen chondrocyte sections were obtained with Keyence BZ-x800 microscope system (Osaka, Japan).

Anti-VPS13D (A304-691A, Bethy Laboratories. Inc), Anti-GFP (AE011, Abclonal), anti-Halo (G9211; Promega), anti-Tubulin (100109-MM05T; Sinobiological), anti-actin (20536-1-AP; Proteintech), anti-VDAC1 (55259-1-AP; Proteintech), anti-TSG101 (A1692; Abclonal) were used at 1:1000 dilutions for western blots. Anti-VPS13D (A304-691A, Bethy Laboratories. Inc) and anti-TSG101 (SC7964; Santa Cruz Biotechnology) antibodies were used 1:100 for immunofluorescence. The following reagents were used in this study: Oleic acid (O1008; sigma); Palmitic acid (P0500; sigma); Doxycycline (1225984; Sigma), BODIPY 493/503 (ThermoFisher Scientific, D3922), BODIPY 558/568 (ThermoFisher Scientific, D2219), NBD-C12 (Abcam, Ab145361), BODIPY 558/568 C12 (ThermoFisher Scientific, D3835), BODIPY FL C16 (ThermoFisher Scientific, D3821), Janilia Fluo® 646 HaloTag® Ligand (GA1120; Promega) and antibiotics such as G418 (10131027) and puromycin (A1113803) were obtained from ThermoFisher Scientific. All EM reagents were purchased from Electron Microscopy Sciences. Lipids were purchased from Avanti Polar Lipids: NBD-PC (810133), NBD-PE (810144), NBD-PS (810198), NBD-PA (810138), NBD-ceramide (810211).

HEK293 cells were pulsed by an incubation in complete medium (DMEM with 10% fetal bovine serum) containing 1 μM BODIPY 558/568 C12 (ThermoFisher Scientific, D3835) for 16 h. Cells were washed with PBS three times and then chased in EBSS for the time indicated.

As described by others^{42 (link)}, mice were injected with 1 μg/g body weight BODIPY™ 558/568 C12 (D3835, Thermo Fisher, MA, USA) via the tail vein, 50 min later, the hearts were collected after removing fat, blood, and auricles and rinsed in PBS. X-ray and fluorescence imaging was performed using a small animal living fluorescence imaging system (IVIS Spectrum, PerkinElmer, USA).

30,000 cells/well were seeded on IBIDI μ-slides (IBIDI, Martinsried, Germany) one day before treatment as indicated (24 h). For antibody staining, cells were washed (PBS), fixed (3% Paraformaldehyde) for 30 min, permeabilized (0.1% Triton-X) and unspecific binding was blocked (5% BSA) after treatment. Primary antibodies (Lamp3/sc-15363, Hsp60/sc-1052, Cox4/4844 NEB, ACADVL/ab118183, ACADM/ab118183, HADHA/ab118183, TFE3/PA5–35210, MITF/sc-71588, TFEB/A303-673A) were applied over night at 4 °C, secondary antibodies (Alexa-Fluor 488/A-11008, Alexa-Fluor 546/A-10040, Alexa-Fluor 633/A-21082) and Hoechst33342 for 45 min at 25 °C. LD were stained by 2 μM Bodipy™ 493/503 for 30 min, fatty acids were tracked by adding 1 μM BODIPY™ 558/568 C₁₂ (both: Thermo Fisher) 16 h prior to end of treatment. Cells were washed, mounted with FluorSave™ Reagent (Beckman Coulter) and covered with a glass coverslip. For life cell imaging 2 μM Bodipy™ 493/503 was added 30 min prior to imaging, 1 μM BODIPY™ 558/568 C₁₂ was added 16 h prior to imaging, 100 nM LysoTracker™ Blue DND-22 or 100 nM MitoTracker™ Green FM were added for 30 min to visualize lysosomes or mitochondria. Medium was exchanged and images were acquired using a Leica TCS SP 8 SMD confocal microscope (Leica TCS SP 8 SMD, Wetzlar, Germany) with a top stage incubator (Oko Lab, Ottaviano, Italy).

To examine directional lipid transfer from adipocytes to ALL cells, 3T3-L1 adipocytes were incubated with 2 µg/ml BODIPY™ 558/568 C₁₂ (4,4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid) (D3835, Thermo Fisher Scientific) for 4 h. The adipocytes were washed with HBSS containing 0.2% fatty acid-free BSA to remove extracellular fatty acids. Subsequently, ALL cell lines at a concentration of 0.2 × 10⁶ cells/ml were cocultured with labelled 3T3-L1 adipocytes for 24 h before being harvested, washed with HBSS containing 0.2% fatty acid-free BSA and analysed by an Amnis® ImageStream®XMk II flow cytometer. Imagining was performed using INSPIRE™ software.