Fatty acid conjugated BODIPY tracer experiments

The sixteen carbon BODIPY FL C16 (Invitrogen, cat# D3821) was used to study fatty acid uptake and intracellular distribution in stable shNDRG1 and shGFP expressing SKBR3 cells (cultured as in lipidomics experiments described above). A 2 mM stock solution was prepared in culture medium supplemented with 0.1% fatty acid free bovine serum albumin (Goldbio cat# A-421-100), and diluted in DMEM/high glucose with L-glutamine, and sodium pyruvate + 10% FBS to achieve the desired concentrations. Cells were seeded at 10,000 viable cells per well, allowed them to adhere for 24 h, and medium was replaced with C16-BODIPY at 100 μM, 50 μM, 25 μM and 12.5 μM in complete DMEM and cells were cultured under normal conditions for 16 h in the presence of the tracer. After 16 h, excess tracer was removed by three consecutive washes in complete DMEM, followed by the fixation protocol described above.

To quantify total tracer uptake, an image analysis routine was established to measure the intensity of cell-sized objects above a background threshold. Cell nuclei were counted, and average C-16 BODIPY signal was expressed as the ratio of overall signal divided by the number of nuclei in each field of view. To quantify lipid-droplet-specific signal, the granule counting and measurement algorithm described above was used. The ratio of lipid-droplet-specific C-16 BODIPY signal to total C-16 BODIPY signal was computed to reflect the flow of tracer fatty acid to lipid droplets.

In MCF7 cells, a protocol to induce lipid droplet formation and live cell tracer incorporation through starvation in Hanks buffered saline solution (HBSS) was developed based on published methods [32]. HBSS consists of 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.63 mM glucose, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, and 4.2 mM NaHCO3. Stable MCF7 cell lines transduced with retroviral full length NDRG1–3X FLAG and retroviral empty vector were plated as described above cultured in ether complete medium or HBSS. For live cell experiments, BODIPY 558/568 C12 (C12-BODIPY) was added to initial culture medium at 100 μM to allow cellular uptake, and followed by extended chase periods. Samples were fixed and stained for lipid droplets as described above. To elaborate the time dependence of the phenomenon and rule out tracer specific effects, lipid droplet formation by cells grown without C12-BODIPY tracer were monitored over the course of 4 days by staining with BODIPY 493/503 using analysis methods described above.