Effect of NCLX expression or activity on glucose dependent cytosolic calcium responses.

<p><b>A.</b> Real time PCR analysis of mRNA NCLX expression normalized to GAPDH in pancreatic primary β cells transfected with siNCLX vs. siControl, n = 3 (*P<0.05). <b>B.</b> Silencing NCLX expression inhibits glucose-induced Ca²⁺ entry in primary β cells. Representative fluorescent traces of cytosolic Ca²⁺ in pancreatic primary β cells transfected with either siNCLX or siControl loaded with Fura 2 AM and stimulated with high glucose following the same experimental paradigm described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g002" target="_blank">Fig. 2A</a>. <b>Insert.</b> Shows representative images of MIN6 cells co-transfected with the Dharmacon siGLO Red transfection reagent. The scale bar is 10 µm. <b>C.</b> NCLX dominant negative construct inhibits glucose dependent cytosolic Ca²⁺ changes in primary β cells. Representative fluorescent traces of primary β cells transfected with dnNCLX or control vector (pcDNA) loaded with Fura 2 AM and treated with high glucose when indicated. <b>D.</b> Averaged rates of cytosolic Ca²⁺ responses of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g004" target="_blank">Fig. 4B</a>, n = 10 (*P<0.05). <b>E.</b> Averaged rates of cytosolic Ca²⁺ responses of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g004" target="_blank">Fig. 4C</a>, n = 10 (*P<0.05). <b>F.</b> Averaged cytosolic Ca²⁺ amplitudes of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g004" target="_blank">Fig. 4B</a>, n = 10 (*P<0.05). <b>G.</b> Averaged cytosolic Ca²⁺ amplitudes of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g004" target="_blank">Fig. 4C</a>, n = 10 (*P<0.05).</p

Effect of NCLX on mitochondrial Ca²⁺ transport, metabolic rate in resting and high glucose dependent manner.

<p><b>A.</b> Knocked down of NCLX modulates mitochondrial calcium transport. Pancreatic primary β cells were infected with lenti-pericam viral particles and transfected with either siNCLX or siControl and superfused with the indicated high glucose Ringer solution. <b>Insert.</b> Representative image of pancreatic primary β cell infected with lenti-pericam. The scale bar is 10 µm. <b>B.</b> Averaged mitochondrial Ca²⁺ influx rates of pancreatic primary β cells of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g005" target="_blank">Fig. 5A</a>, n = 3 (*P<0.05). <b>C.</b> Averaged mitochondrial Ca²⁺ efflux rates of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g005" target="_blank">Fig. 5A</a>, n = 3 (*P<0.05). <b>D.</b> Effect of NCLX on respiratory chain activity determined by monitoring NAD(P)H intrinsic fluorescence in pancreatic primary β cells, transfected with either siNCLX or siControl before and after application of high glucose Ringer solution. FCCP or high glucose Ringer's solution was added where indicated.</p

NCLX is expressed in mitochondria of pancreatic β cells and mediates mitochondrial Ca²⁺ transport.

<p><b>A</b>. Immunoblot analysis of NCLX expression in total lysate and isolated mitochondria in MIN6 cells (20 µg). <b>B.</b> Immunoblot analysis of NCLX expression in siNCLX vs. siControl (20 µg) transfected MIN6 cell lysates. VDAC and β Actin were used as mitochondrial and cytosolic markers, respectively. <b>C.</b> Knock down of NCLX expression increases Ca²⁺ influx and inhibits mitochondrial Ca²⁺ efflux. At the indicated time, cells were superfused with high K⁺ Ringer solution while monitoring mitochondrial Ca²⁺ in MIN6 cells transfected with mito-pericam and either siNCLX or siControl. <b>D.</b> Dominant negative NCLX construct increases Ca²⁺ influx and inhibits mitochondrial Ca²⁺ efflux. Representative fluorescent traces of pancreatic MIN6 cells co-transfected with mito-pericam and either dnNCLX or control vector (pcDNA), while applying the same experimental paradigm described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1C</a>. <b>Insert.</b> Representative images of MIN6 cells co-transfected with mito-pericam. The scale bar is 10 µm. <b>E.</b> Averaged rates of mitochondrial Ca²⁺ influx of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1C</a>, D, n = 9 (*P<0.05). <b>F.</b> Averaged rates of mitochondrial Ca²⁺ efflux of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1C</a>, D, n = 9 (*P<0.05). <b>G.</b> Silencing of NCLX expression inhibits mitochondrial Ca²⁺ efflux following a metabotropic cytosolic Ca²⁺ response. Cells were co-transfected with mito-pericam and either siNCLX or siControl and superfused with Ca²⁺ free Ringer solution containing 50 µM ATP, while monitoring the Ca²⁺ response. <b>H.</b> Averaged rates of mitochondrial Ca²⁺ influx of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1G</a>, n = 7 (*P<0.05). <b>I.</b> Averaged rates of mitochondrial Ca²⁺ efflux of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1G</a>, n = 7 (*P<0.05).</p

Effect of NCLX silencing expression on ATP production and insulin secretion.

<p><b>A.</b> Effect of NCLX silencing expression on ATP production. The ATP content was determined in pancreatic primary β cells lysates transfected with either siNCLX or siControl and stimulated with high glucose in the indicated times (<i>see Experimental Procedures</i>), n = 3 (*P<0.05). <b>B.</b> Effect of NCLX knocked down expression on glucose dependent insulin secretion. Cultured pancreatic primary β cells were transfected with either siNCLX or siControl and amounts of secreted insulin were determined in the indicated times, n = 3 (*P<0.05).</p

Role of NCLX in cytosolic Ca²⁺ responses.

<p><b>A.</b> Silencing of NCLX expression inhibits cytosolic Ca²⁺ responses. MIN6 cells either transfected with siNCLX or siControl were loaded with Fura 2 AM and depolarized with high K⁺ Ringer solution, while monitoring cytosolic Ca²⁺ responses. <b>B.</b> Dominant negative mutant NCLX, dnNCLX inhibits cytosolic Ca²⁺ responses. MIN6 cells transfected with either dnNCLX or control vector (pcDNA) were loaded with Fura 2 AM and cytosolic Ca²⁺ was monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g003" target="_blank">Fig. 3A</a>. <b>C.</b> Averaged rates of cytosolic Ca²⁺ responses of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g003" target="_blank">Fig. 3A</a>, n = 12 (*P<0.05). <b>D.</b> Averaged rates of cytosolic Ca²⁺ responses of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g003" target="_blank">Fig. 3B</a>, n = 12 (*P<0.05). <b>E.</b> Averaged cytosolic Ca²⁺ response amplitude of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g003" target="_blank">Fig. 3A</a>, n = 12 (*P<0.05). <b>F.</b> Averaged cytosolic Ca²⁺ response amplitude of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g003" target="_blank">Fig. 3B</a>, n = 12 (*P<0.05).</p

NCLX mediates glucose dependent mitochondrial Ca²⁺ transport and modulates the basal mitochondrial membrane potential and calcium resting levels.

<p><b>A.</b> Silencing of NCLX expression blocks the glucose dependent mitochondrial Ca²⁺ efflux. The mitochondrial Ca²⁺ transport was monitored in MIN6 cells co-transfected with mito-pericam and either siNCLX or siControl. Cells were first superfused with low glucose (3 mM) Ringer followed by high glucose (20 mM) Ringer solution. <b>B.</b> Averaged rates of mitochondrial Ca²⁺ influx of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g002" target="_blank">Fig. 2A</a>, n = 11 (*P<0.05). <b>C.</b> Averaged rates of mitochondrial Ca²⁺ efflux of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g002" target="_blank">Fig. 2A</a>, n = 11 (*P<0.05). <b>D.</b> Silencing NCLX modulates the basal but not glucose dependent change in mitochondrial membrane potential. Changes in mitochondrial membrane potential were monitored in MIN6 cells transfected with siNCLX or siControl, superfused continuously with 0.05 µM TMRM. FCCP 5 µM was added in the indicated times to calibrate the signal. <b>E.</b> Effect of knock down of NCLX expression on mitochondrial resting Ca²⁺ in MIN6 cells transfected with siNCLX vs. siControl. Averaged mitochondrial Ca²⁺ basal signals, n = 10 (*P<0.05).</p