Mitophagy proteins translocate to mitochondria and are necessary for T3 stimulation of mitophagy. a Immunoblot showing mitochondrial protein ubiquitination and localization of ULK1, p62, LC3-II, and Drp1 proteins in isolated mitochondrial fraction from T3 (100 nM/48 h)-treated TRβ1-HepG2 cells. Purity/enrichment of the mitochondrial fraction (Mito) was verified by the absence of β-Tubulin (cytosolic) and LAMP-1 (lysosomal) relative to its level in the whole cell lysate (WCL) for the same amount of VDAC levels. b TRβ-HepG2 cells transiently expressing Mito-mRFP-EGFP were treated with 100 nM T3 for 48 h with or without ULK1 KD followed by visualization using confocal microscopy (40× magnification). Nuclei were stained with DAPI (blue). In the images, fluorescence signals indicate the expression of Mito-mRFP-EGFP targeting mitochondria: yellow color no mitophagy, red color mitophagy. c Quantitative analysis of the RFP (red) fluorescence to denote % mitophagy. Quantification of images (at least 10 transfected cells per each sample in 3 different fields) was conducted with ImageJ software. Bars represent the mean of the respective individual ratios ± SD (*p < 0.05).