Fig 2: Supplementary Figure 1. Localization of PLIN1 exclusively to lipid droplets under basal and stimulation conditions. Brown adipocytes induced to express EYFP-SRC1 and PLIN1-PPARa, were treated with vehicle control (Basal) or stimulated for 20 min (1 µM Iso), washed with PBS and fixed with 4% paraformaldehyde. Cells were then permeabilized, stained, and imaged for PLIN1 (pseudocolored red), nuclei (DAPI, pseudocolored blue) and EYFP-SRC1 (pseudocolored green). Merged images (PLIN1 + EYFP-SRC1) show that PLIN1 is localized exclusively to lipid droplets under basal and stimulated conditions and does not localize to the nucleus.

Fig 3: Design of a fluorescent sensor to detect fatty acid trafficking on lipid droplets and in the nucleus. (A) Schematic for the design of a doxycycline (Dox) inducible bipartite fluorescent sensor to detect fatty acids. The co-activation domain of SRC1 is tagged to EYFP (EYFP-SRC1), separated by a P2A self-cleaving site with PLIN1-PPARa fusion, which targets the ligand binding domain of PPARa to lipid droplets. Dox binds reverse tetracycline-controlled transactivator (rtTA3), which promotes binding to the TRE2 promoter to induce expression of the fatty acid sensor. (B) In the presence of ligand (fatty acid; FFA), EYFP-SRC1 binds to PLIN1-PPARa on the surface of LDs. EYFP-SRC1 can also translocate to endogenous nuclear receptors that are sensitive to fatty acids. Stable expression of the fluorescent fatty acid reporter in brown adipocytes under basal conditions (C) and protein kinase A (PKA) stimulation (Stim; forskolin/IBMX) for 20 min (D). Arrows note accumulation of EYFP-SRC1 on lipid droplets while arrowheads note accumulation within the nucleus following stimulation. Quantification of the region of interest on lipid droplet (E) and nucleus (F) normalized to the cytosol signal and expressed as fold of basal.