MitoTracker, LipidTOX, and UCP1 staining.

Preadipocytes were plated and differentiated on 4-well glass slides (Millicell EZ SLIDES, MilliporeSigma). For visualization of mitochondria, live cells were washed with warm media and incubated with MitoTracker Red CM-H2XRos (250 nM) for 20 minutes, then fixed with 4% paraformaldehyde. For staining of lipid droplets, fixed cells were incubated with HCS LipidTOX Green (1:200) for 30 minutes at room temperature after permeabilization with 0.1% Triton X-100 for 5 minutes. Subsequently, cells were incubated overnight with UCP1 (ab155117, Abcam) (1:250), washed in PBS, and incubated with the relevant secondary antibody at room temperature for 1 hour. After the final washes, antifade mounting medium with DAPI (VECTASHIELD HardSet, Vector Laboratories, Maravai LifeSciences) was added to the slides, and images were taken the following day. Image acquisition was performed with an upright Zeiss Axio Observer Z1 microscope using Zen software (2012; Zeiss). Images were minimally processed to adjust brightness and contrast.