Immunohistochemical staining for PV (red) and EGFP (green) in the kidney of wildtype (WT) or PV-deficient (PV-/-) mice.

<p>In WT (PV-EGFP) kidney, the expression of EGFP (upper panel) and PV (middle panel) is restricted to DCT1 and shows 100% colocalization (lower panel). The qualitative distribution of EGFP is the same also in EGFP-PV PV-/- mice, however endogenous PV is completely absent.</p

Primers used for genotyping and qRT-PCR.

<p>Primers used for genotyping and qRT-PCR.</p

Western blot of COX1 and ATP synthase subunit β protein expression after PV silencing via shPvalb.

<p>Down-regulation of PV induced a significant rise in COX1 expression (*p = 0.00002, n = 4 independent experiments, mean ± sem), ATP synthase subunit β expression was unchanged (n = 3 independent experiments, mean ± sem; n.s.).</p

Relative mRNA expression levels of 7 selected genes determined by qRT-PCR based on results from Gene Chip Analysis (for more details, see S1 Table).

<p>RNA levels of genes implicated in mitochondrial Ca²⁺ regulation: <i>Efhd1</i>, <i>Micu1</i>, <i>Mcu</i>, <i>Mcur1</i> as well as other mitochondrial genes (<i>Ucp2</i>, <i>COX1</i> and <i>Atp5b</i>) are upregulated in PV-deficient DCT cells (results are from n = 4 animals per genotype, mean ± sem. The upregulation is statistically significant for <i>Micu1</i> and <i>Atp5b</i> (p<0.05, Mann-Whitney U test).</p

Evaluation of the relative mitochondrial volume in MDCK cells by FACS.

<p>A) The normalized histogram showed a left shift for the PV-positive MDCK PV15 (light green peak) compared to control MDCK cells (orange peak), the autofluorescence of the 2 cell lines was indistinguishable (overlapping dark green peaks). B) All PV-expressing MDCK clones exhibited a statistically significant reduction in the mean fluorescence intensity (n ≥ 2 experiments, mean ± sd). One-way ANOVA analysis resulted in a highly significant overall p-value (p = 7.23 x 10⁻¹⁸), indicative of differences between groups; within the group of PV-expressing clones no statistical differences were detected (p = 0.1589). A comparison of each PV clone with the control MDCK cells showed significant differences (t-test; p<0.05 for all PV clones).</p

Silencing of ectopic PV in MDCK cells (clone PV15).

<p>A) Down-regulation of <i>Pvalb</i> mRNA via constitutive lentiviral-mediated shRNA decreases PV protein expression levels after 10 days of puromycin selection. B) PV down-regulation is less potent via the IPTG-inducible shRNA MISSION^® system tested in the PV-positive MDCK clone PV15. C) IHC of PV clone PV15 infected with constitutive lentiviral shPvalb after 10 days of puromycin selection (right image) in comparison to untreated cells (left image).</p

CCCP-induced changes in the mitochondrial membrane potential evaluated by two fluorescent lipophilic cationic dyes: Mitotracker Red (MTR)/Green (MTG) (A) and JC-1 (B).

<p>Treatment with the uncoupler CCCP decreased the mitochondrial membrane potential. The concentration dependent decrease was bigger in the PV-expressing MDCK PV15 cells compared to control MDCK cells (n = 3 independent experiments, mean ± sem, differences between control and PV15 were statistically significant at all CCCP concentrations (pairwise t-test; p<0.05 marked with (*) for all pairs in (A) and (B)). C) The cuvette measurements (A,B) were confirmed by qualitative fluorescence microscopy. Collapsing of the mitochondrial membrane potential by CCCP reduced MTR fluorescence emission in both control (upper row) and PV15 MDCK (lower row) cells; the decrease was more pronounced in the PV-expressing PV15 clone.</p

Western blots for COX1 and ATP synthase subunit β in DCT lysates from 4 control (WT) and 4 PV-/- mice.

<p>Values are mean ± sem of 3 independent experiments. In PV-/- mice the expression of COX1 was augmented by 31.2 ± 7.0% compared to WT (*p = 0.03706), while no changes were observed for ATP synthase (n.s.). Representative Western blot signals are shown in the upper part.</p

Madin-Darby canine kidney (MDCK) cells stably expressing PV.

<p>Lentiviral infection of MDCK cells led to cytoplasmic expression of PV evidenced by PV immunohistochemistry; PV expression levels were variable in clones selected by serial dilutions (A). Relative PV protein expression levels of selected clones were quantified by Western blot analyses (B). Non-transfected (Con) and EGFP-transfected clones (EGFP2) were negative for PV. Expression levels in individual clones were considerably different; e.g. levels in clone PV11 were almost 10-fold lower than in the high-expressing clone PV15. COX1 and ATP synthase subunit β protein levels of clones without PV and clones highly expressing PV (PV15, PV19, PV29) were compared (C). COX1 was significantly reduced in the PV-expressing clones (*p = 0.0099), ATP synthase subunit β levels were not different; p = 0.890823; n = 6 independent experiments, mean ± sem). The mRNA level of <i>COX1</i> gene coding for COX1 was assessed by qRT-PCR (D). In the PV-expressing PV15 clone, the <i>COX1</i> signal was decreased by 48%; p = 0.00005). The results are the mean of 2 independent experiments (mean ± sem).</p

Volumetric analysis of mitochondrial content in WT and PV-transfected C2C12 cells.

<p>A–C: Confocal images of WT C2C12 cell loaded with Mito Tracker Red (A), Calcein-AM (B), DAPI (C): x–y image Z-stack projection (large image), x–z (bottom) and y-z (right) image. Scale bar is 40 µm. These images were transferred to Imaris 3D to calculate relative mitochondrial volume. D–F: Bar histographs of relative mitochondrial volume (D), cytoplasmic volume (E) and relative nuclear volume (F) in C2C12 WT (light bars) and PV-clones (dark bars) either in control medium (left bars) or incubated for 48 h with Br-A23187 (1 µM; right bars). D) The relative mitochondrial volume was increased in WT C2C12 after ionophore treatment (*; p<0.05). The mitochondrial volume in PV-clones, both in control medium or after 48 h ionophore treatment was lower than in untreated WT C2C12 cells; * p<0.05 vs. WT; n>30 cells). The decrease in mitochondrial volume in PV-clones after ionophore treatment was not significant (n.s.) E) No differences in cytoplasmic volumes were detected. F) The nuclear volume in ionophore-treated WT C2C12 was slightly larger than in untreated WT cells (*; p<0.05).</p