Figure 4.

Permeabilized INS1E cells lack or show minimal Complex l-dependent respiration: A) Rotenone-sensitive respiration in INS1E v/s. astrocytes. Primary rat astrocytes were used as positive homologous controls. Respiration was assessed in Ca²⁺-free LKB containing 16.7 mM glucose. Injections- PFO: 1 nM rPFO; GMA/R: 10 mM gutamate and 10 mM malate (GM) with 1 mM ADP in the presence (INS1 E_GM+Rot, HEK293_GM+Rot) or absence of 1 μM rotenone (INS1E_GM, HEK293_GM). B) Relative Complex I to Ill-dependent respirations in INS1E cells. Respiration medium was the same as in panel A. Injections- Subs+ADP: 1 nM rPFO and 1 mM ADP were added with substrates supporting Complex I- (10 mM glutamate and 10 mM malate, G+M), Complex II- (10 mM succinate, Succ), Complex III- (10 mM glycerol-3-phosphate, G-3-P)-dependent respiration. C) Complex I, II and Ill-dependent respirations in intact INS1E cells measured using specific inhibitors, the rotenone (1μ M), TTFA (400μM) and antimycin-A (4μg/ml) respectively. D) ln-gel activity of Complex I. NADH- dehydrogenase activity was assayed following BN-PAGE. Mitochondria isolated from the INS1E cells starved for indicated time periods were used for BN-PAGE analyses. HEK293 mitochondria served as positive controls. Data mean ± SD (n= at least 3 wells/group) are shown in panels A & B. They are representative of >3 independent replicates.