Figure 3
SOCS6 regulates mitochondrial morphology. (a) Morphology of mitochondria stained with MitoTracker in AD293 cells transiently transfected with pcDNA3, pcDNA3-SOCS6, or pcDNA3-SOCS3 (green, anti-HA). Scale bar: 10 μm. (b) AD293 cells were transfected with pcDNA3-SOCS6, and immunofluorescence of HA-SOCS6 was performed after labeling cells with MitoTracker. SOCS6-expressing cells were scored for tubular or fragmented mitochondrial morphology at the designated time points (left panel). Cells in 24-well plates were transfected with increasing amounts (0.04, 0.12, and 0.4 μg) of pcDNA3-SOCS6, and mitochondrial morphology in SOCS6-expressing cells was determined at 24 h (right panel). A total of 300 SOCS6-expressing cells were examined for each condition in the experiments and data were derived from three independent experiments. (c) Confocal immunofluorescence of SOCS6 (anti-SOCS6) in the control-KD and SOCS6-KD HeLa cells. Scale bar: 10 μm. (d and e) Micro-P analysis of mitochondrial morphology in HA-SOCS6-expressing AD293 (d) and SOCS6-KD HeLa (e) cells. The Micro-P algorithm classified mitochondria into small fragmented mitochondrion (type 1, blue), large fragmented mitochondrion (type 2, yellow), curved, branched, or circular tubular mitochondrion (type 3, red) and tubular mitochondrion (type 4, green). A representative presentation of Micro-P analysis is shown at the top. Bar graphs at the bottom show the percentages of type 1 to type 4 mitochondria (left panel) and the length and width of type 3 and type 4 mitochondria (right panel) in 300 cells, and are expressed as the means±S.E.M. **P<0.01 and ***P<0.001 by Student’s t test. (f) Electron micrographs of mitochondria in AD293 cells transiently transfected with pcDNA3 and pcDNA3-SOCS6 (left panel) and HeLa cells stably harboring the control shRNA-Luc or shRNA-SOCS6 (right panel). Scale bars: 0.5 μm. Quantitative analysis of intramitochondrial ultrastructure was performed by measuring CM and IBM length using ImageJ software and the ratio of CM/IBM is shown. A total of 15 mitochondria from each category were scored
