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. 2015 Jun 4;112(24):E3104–E3113. doi: 10.1073/pnas.1421290112

Fig. 6.

Fig. 6.

Mitochondrial function is transiently decreased by hypertonic and Cl− ionophore challenge. (A and B) [ATP]i in LLC-PK1 cells was measured with ATP indicator ATeam (A, dark colors) or fluorometric analysis (B) before and after (arrow) NaCl or urea challenge [400 (A) or 500 (B) mOsmol/kg] and oligomycin (10 µg/mL)/2-DOG (10 mM). (C) [ATP]i in LLC-PK1 cells was measured by ATeam (dark tone) before and after the addition of TBTN followed by oligomycin/2-DOG. In A, to estimate the actual relative loss of ATP, the wild-type ATeam FRET curves are normalized to the ATP-insensitive mutant probe FRET curves to account for volume-induced, ATP-independent FRET signals. Mutant probe responses are illustrated in light tones (A and C) on the left axes. (D) Decreased FITC-dextran–loaded endosome motility kinetics (black, right axis) is similar to that of ATeam FRET signal loss (gray, left axis) in LLC-PK1 cells after addition of oligomycin/2-DOG (arrow). Error bars show mean ± SEM from four independent experiments. (E) Time-lapse pseudocolored images of GFP-α-tubulin and GFP-β-actin in LLC-PK1 cells before and after the addition of oligomycin/2-DOG illustrate decreased motility in both microfilaments after challenge. (Scale bars: 10 µm.) Representative images are shown on the left, and quantification of motility in three independent experiments is shown on the right. (F) Ratiometric imaging of the mitochondrial membrane potential dye JC-1 (7.7 µM) in control (Ctl) and LLC-PK1 cells challenged with NaCl (500 mOsmol/kg), Cl− ionophores (TBTN), or the positive controls CCCP (1 µM) or ArA (20 µM) for 15 min. Images show JC-1 fluorescence in control cells and after 10 min of NaCl challenge. (G) Analysis of mitochondrial fission in LLC-PK1 cells pretreated with MitoTracker Red (500 nM) and then challenged with NaCl (500 mOsmol/kg) or ionophores (TBTN, 10 min) or with CCCP or ArA (15 min). Error bars show mean ± SEM from four independent experiments. *P < 0.05 vs. control. Images show MitoTracker fluorescence in control cells and after 10 min of NaCl challenge. (Scale bars: 10 µm.)