Figure 5
SOCS6 impairs mitochondrial function. (a) PEG cell fusion assay. Two HeLa cell clones each containing mitochondrial-targeted red fluorescent protein from Discosoma (DsRed-mito) or EYFP (EYFP-mito) were transiently transfected with the pcDNA3 or pcDNA3-SOCS6. After PEG treatment, confocal images were taken at the indicated time points. Scale bar: 10 μm. (b) FRAP analysis. HeLa cells were transiently transfected with pIRES-EYFP (mock) or pIRES-EYFP-SOCS6 (SOCS6), and loaded with MitoTracker. Photobleaching of MitoTracker fluorescence was performed in ROIs (indicated in white rectangles). Fluorescence images were taken and enlarged images within the blue squares of the prebleached, bleached, and recovered are shown after 30, 60, and 180 s. Scale bar: 10 μm. Relative fluorescence intensity of MitoTracker in ROIs was recorded and plotted during a photobleaching protocol as a function of time (n=20 cells). Error bars represent the S.E.M. ***P<0.001 by Student’s t test. (c–f) AD293 cells transfected with pcDNA3 (mock) or pcDNA3-SOCS6 (SOCS6) (left panels), and HeLa cells stably harboring lentivirus-based shRNA targeting luciferase or SOCS6 (right panels) were measured for mitochondrial membrane potential (Δψm) using the cationic dye JC-1 by flow cytometry (c), total cellular ATP level (d), lactate production (e), and glucose uptake (f). Percentage shown relative to the control cells were derived from three separate experiments and are presented as the means±S.E.M. ***P<0.001 by Student’s t-test. (g and h) The mock- and HA-SOCS6-transfected HeLa cells and the control-KD and SOCS6-KD HeLa cells were subjected to real-time measurement of OCR (g) and ECAR (h) using an Extracellular Flux Analyzer. OCR was measured at steady state followed by sequential incubation with 3 μM oligomycin, 1 μM FCCP, and 5 μM antimycin A. ECAR was measured at steady state followed by sequential incubation with 25 μM glucose, 3 μM oligomycin, and 50 μM 2-DG. Data were derived from three separate experiments and are presented as the means±S.E.M. *P<0.05, and **P<0.01 by Student's t-test. (i) Total cell lysates were prepared and subjected to immunoblotting against the indicated proteins
