Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex - Fig 4

<p>Severe EAE increased the cytosolic MnSOD protein abundance (A). Both mild and severe EAE elevated the mitochondrial MnSOD protein levels (B). Severe EAE increased the total activities of cytosolic and mitochondrial MnSOD (C). EAE had no significant effect on the specific activity of cytosolic MnSOD (D), but reduced the specific activity of mitochondrial MnSOD (E). The cytosolic and mitochondrial proteins were separated by centrifugation and analyzed by Western analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g001" target="_blank">Fig 1C</a>. Total MnSOD activity was measured by the difference of absorption at A₄₅₀ between heat-inactivated and live extracts. The readings were normalized to the reading from the first mouse in the control group. The specific MnSOD activity is expressed by the total MnSOD activity normalized to MnSOD protein abundance. (*p < 0.05 vs Control, unpaired <i>t</i> test except for C by One-way ANOVA).</p

Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex - Fig 2

<p>Severe EAE increased production of ROS from the isolated mitochondria in the mouse renal cortex (A) and in the native renal cortex (D). Mitochondrial ROS production was measured by increases of fluorescence from dihydroethidium (A). The same dye (50 mg/kg) was injected intraperitoneally. The frozen sections of the kidney cortex were imaged 18 hours after injection, representatives of two independent experiments (B to D) (*p < 0.05 vs Control, One-way ANOVA, the sample sizes are same to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g001" target="_blank">Fig 1</a>).</p

Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex - Fig 9

<p>Catalase reduced the monensin-induced increases of Na,K-ATPase activity (A, n = 4) and the cytosolic Na,K-ATPase α1-subunit protein abundance (B, n = 4). The Na,K-ATPase activity was measured by ouabain-sensitive release of inorganic phosphate, which reacted with malachite green and was quantified by increases in the absorption at A₆₃₀. Membranes were probed with antibodies against aldose reductase (AR, cytosolic marker) and cytochrome c oxidase subunit IV (COX4, mitochondrial marker) to show adequate separation of these two compartments and with actin antibody to show roughly equal loading. (*p < 0.05 vs Control, **p < 0.05 vs Monensin, Two-way ANOVA, n = 4).</p

Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex - Fig 5

<p>Monensin (10 μM) increased the mitochondrial complex II activity (A, n = 5), but decreased the complex I activity (B, n = 5) and had no significant effect on the complex IV activity (C, n = 4). Ouabain (4 nM) blocked the effect of monensin on the complex II activity (A). Monensin had no significant effect on the cytosolic ATP level, but decreased the mitochondrial ATP level (D, n = 6). HEK293 cells were pre-incubated with ouabain for 60 min and then monensin was added for 24 hours before they were collected. The complex I, II and IV activities were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g001" target="_blank">Fig 1A and 1B</a>. ATP level was measured by a luminescence-based ATP Determination Kit from Molecular Probes. (*p < 0.05 vs Control, **p < 0.05 vs Monensin Two-way ANOVA).</p

Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex - Fig 6

<p>Monensin increased the mitochondrial ROS in the isolated mitochondria (A, n = 5) and in live HEK293 cells (B, n = 4 and C). Ouabain inhibited the effect of monensin. (A) HEK293 cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g005" target="_blank">Fig 5</a> and ROS from isolated mitochondria were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g002" target="_blank">Fig 2A</a>. B is average of 4 independent flow cytometry analyses of mitochondrial ROS in live HEK293 cells. MFI: mean fluorescence intensity. C is a representative flow cytometry analysis. After HEK293 cells were incubated with 4 nM ouabain for 60 min, monensin (10 μM) was added into the medium and the cells were incubated for an additional 60 min, then Mito-sox Red (2.5 μM) was added and the cells were incubated for another 60 min before they were collected for analysis. (*p < 0.05 vs Control, **p < 0.05 vs Monensin, Two-way ANOVA).</p

Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex - Fig 7

<p>Monensin elevated the mitochondrial MnSOD protein level without a significant effect on the cytosolic MnSOD protein abundance (A, n = 5). Ouabain (4 nM) inhibited the effect of monensin on the mitochondrial MnSOD protein (B, n = 5). Membranes were probed with antibodies against aldose reductase (AR, cytosolic marker) and cytochrome c oxidase subunit IV (COX4, mitochondrial marker) to show adequate separation of these two compartments and with actin antibody to show roughly equal loading. Monensin had no significant effect on the total activity of cytosolic or mitochondrial MnSOD (C, n = 5), but significantly reduced the specific activity of mitochondrial MnSOD and this effect was blocked by ouabain (D, n = 5). HEK293 cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g005" target="_blank">Fig 5</a>. The total and specific MnSOD activities were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g004" target="_blank">Fig 4</a>, but the total and specific activities were normalized to the control in each experiment. (*p < 0.05 vs Control or Monensin plus Ouabain in D, **p < 0.05 vs Monensin, One-way ANOVA for A, Two-way ANOVA for B and D).</p

Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex - Fig 1

<p>EAE increased the activities of mitochondrial complex II and IV, but not complex I from the mouse renal cortex (A and B). Severe EAE had a trajectory to further augment the complex IV activity over mild EAE. This effect was associated with an increase of COX4 protein level (B and C). The activities of complex I and II were measured by the difference of absorption at A₆₃₀ between the assay buffer alone and assay buffer with 5 μg mitochondria extracts (A). The complex IV activity was assayed by increases in absorption at A₅₆₂ in the presence of 5 μg mitochondria extracts (B). The protein extracts from mouse kidney cortex were separated in a 4–12% Bis-Tris gel (Invitrogen) and probed with antibodies (C). (*p < 0.05 vs Control, p = 0.065 vs mild EAE; Control n = 7, Mild EAE n = 5, Severe EAE n = 6; One-way ANOVA for A and B, unpaired <i>t</i> test for C).</p

Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex - Fig 8

<p>Catalase reduced the monensin-induced increases of mitochondrial ROS in the isolated mitochondria (A) and in live HEK293 cells (B, n = 5 and C, n = 5). Mitochondrial ROS were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g006" target="_blank">Fig 6</a> except ouabain was replaced by catalase (400U/ml) (*p < 0.05 vs Control, **p < 0.05 vs Monensin, Two-way ANOVA).</p

Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex - Fig 10

<p>Catalase reduced the monensin-induced increases of complex II activity (A, n = 5) and cytosolic ATP level in the presence of monensin (B, n = 6). Monensin reduced the mitochondrial ATP level and complex I activity, and catalase did not significantly affect the effects of monensin (B and C, n = 4). The complex I and II activities and ATP levels were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g005" target="_blank">Fig 5</a> except ouabain was replaced by catalase (400 U/ml). (*p < 0.05 vs Control, **p < 0.05 vs monensin, Two-way ANOVA).</p

Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex - Fig 11

<p>Catalase reduced the monensin-induced increase of mitochondrial MnSOD protein abundance (A, n = 5). The same membrane used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g009" target="_blank">Fig 9A</a> was incubated with the antibodies against MnSOD, AR, COX4 and actin (A). Catalase had no significant effect on the total MnSOD activity (B, n = 5), but reversed the monensin-induced inhibition of the specific MnSOD activity (C, n = 5). Knockdown of MnSOD reduced cellular ATP level with or without monensin (D, n = 4). The protein levels, total and specific activities of MnSOD were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g007" target="_blank">Fig 7</a>. HEK293 cells were transfected with 80 nM control or MnSOD siRNA overnight and treated with or without monensin for 24 hours. The cellular ATP levels were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196277#pone.0196277.g005" target="_blank">Fig 5</a>. (*p < 0.05 vs Control or Monensin plus Catalase in C, **p < 0.05 vs Monensin in A and control siRNA plus ethanol in D, Two-way ANOVA).</p