Fig 5.

AZT and d4T induce accumulation of dysfunctional mitochondria and increased ROS production. 3T3-F442A cells were incubated in the absence or presence of AZT (6, 30, and 150 μM), d4T (3, 15, and 75 μM), or 3TC (8, 40, and 200 μM) for up to 6 days. (A) Flow cytometry analysis of total mitochondrial mass using MitoTracker Green (MTR Green). (B) 3T3-F442A cells transduced with lentiviral particles expressing shATG5 or a nonspecific shRNA control (shC). Flow cytometry analysis of total mitochondrial mass using MTR green (left) with representative histogram (right) immediately after puromycin selection. (C) Flow cytometry analysis of ROS production of 3T3-F442A cells incubated in the absence or presence of AZT (6 and 150 μM), d4T (3 and 75 μM), or 3TC (8 and 200 μM) for 72 h performed using chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H₂DCFDA). (D) 3T3-F442A cells transduced with lentiviral particles expressing shATG5 or a nonspecific shRNA control. Flow cytometry analysis of ROS production using CM-H₂DCFDA immediately after puromycin selection (left), with representative histogram (right). Different scaling between panels A and B as well as between panels C and D resulted from different settings in flow cytometry experiments. (E) 3T3-F442A cells were incubated with drugs as described for panel A or inhibitors of autophagosome formation as indicated in Fig. 4D, and Oil red O staining at the end of the adipogenic conversion was quantified in comparison to control results. (F) 3T3-F442A cells transduced with lentiviral particles expressing shATG5 or a nonspecific shRNA control were induced to differentiate, and Oil red O staining was quantified at the end of the adipogenic conversion. All data are presented as means ± SD and are representative of the results of at least three independent experiments with three to eight replicates. *, P < 0.05; **, P < 0.01.