T3 increases autophagy and mitophagy in hepatic cells. a Model showing how mitochondria-specific mRFP-GFP protein detects mitophagy. b Monitoring mitophagic flux using dual fluorescence p-mito-mRFP-EGFP reporter (pAT016) in HepG2 cells. Lysosomal delivery of the tandem fusion protein Mito-mRFP-EGFP along with entire mitochondria results in differential quenching and degradation of the two individual fluorochromes, thus allowing for visual analysis of mitophagic flux. TRβ1-HepG2 cells transiently expressing Mito-mRFP-EGFP were treated with 1 nM or 100 nM T3 for 48 h followed by visualization using confocal microscopy (40× magnification). Nuclei were stained with DAPI (blue). In the images, fluorescence signals indicates the expression of Mito-mRFP-EGFP targeting mitochondria: yellow color no mitophagy or normal cytosolic mitochondria, red color mitophagy or mitochondria inside lysosomes. c Quantitative analysis of the RFP (red-only) fluorescence to denote % mitophagy was done. Quantification of images (at least 20 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). d Electron micrograph of primary mouse hepatocytes treated with T3. EM of untreated control and T3-treated (100 nM/24 h) mouse hepatocytes showing increased mitophagy (Denoted by arrows showing autophagosomes containing mitochondria) under T3 treatment. Scale bar 1 µm and in enlarged figures are 0.2 µm. e
Bar graphs showing % of autophagosomes (AVs) containing mitochondria in control and T3-treated primary mouse hepatocytes based on EM micrograph images. Scoring was done by counting 10–15 different autophagic vesicles in 5 random fields per condition (n = 3, *p < 0.05.