Fig 1: Quantitative assessment of mitochondrial morphology and function in ciPTEC-OAT1 and AAC3-/-. ciPTEC-OAT1 (black), AAC3-/--1 (dark grey) and AAC3-/--2 (light grey) were matured and evaluated on a functional and morphological level (A). Citrate synthase (CS) activity was determined (B). In addition, cells were evaluated by fluorescence microscopic assessment using 25 nM tetramethylrhodamine methyl ester (TMRM), as described before (Iannetti et al. 2016). Co-staining with Hoechst was performed to correct for cell count. The number of mitochondria per cell (C), mitochondrial size (D), total mitochondrial area per cell (E), mitochondrial membrane potential (F), aspect ratio (a measure of mitochondrial length: ratio between major and minor axis) (G) and mitochondrial roundness (measure of mitochondrial length and degree of branching) (H) were evaluated. Significance was determined by one-way ANOVA with Dunnett’s post hoc analysis, *p < 0.05 and **p < 0.01, mean ± SEM, N = 3 independent experiments
Fig 2: Development and validation of CRISPR/Cas9-mediated knockout of AAC3 in ciPTEC cells. Quantitative PCR was applied to assess the mRNA expression levels of the four human isoforms of AAC in conditionally immortalized proximal tubule epithelial cells (ciPTEC), before (A) and after (B) application of CRISPR/Cas9, for wild-type ciPTEC-OAT1 (black), AAC3-/--1 (dark grey) and AAC3-/--2 (light grey), relative to GAPDH expression. Two independently generated AAC3 knockout cell lines were included in the experiments. In addition, protein levels of AAC1, AAC2 and AAC3 were determined by Western Blot (C) in which COX5A was used as loading control. AAC4 was excluded from analysis as qPCR revealed no mRNA expression, also in line with expression patterns described before (Dolce et al. 2005; Jang et al. 2008; Kunji et al. 2016b; Stepien et al. 1992). Functional AAC-mediated transport of ADP (D) and ATP (E) was investigated, using radioactivity and bioluminescence. Mitochondrial respiration was assessed by the mito stress test (Agilent), as described by the manufacturer, performed in culture medium containing 10 mM glucose, using the Seahorse XF Analyzer to evaluate oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) of the different mitochondrial complexes (F and G), upon following injection of complex specific inhibitors oligomycin A (2.5 µM, complex V), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 1 µM) and rotenone/ antimycin A (1 µM/2.5 µM). Basal respiration (orange) consists of respiratory capacities under resting conditions, maximal respiration (green) is the maximal respiratory capacity of a cell. Spare capacity (red) was determined by subtracting basal respiration from maximal respiration (F and H). Cellular ATP production rate was assessed by the ATP rate assay, using the Seahorse XF Analyzer (I). Data was corrected for cell count, assessed by fluorescence microscopy after Hoechst staining. Significance was determined by one-way ANOVA with Dunnett’s post hoc analysis, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, mean ± SEM, N = 3 or 4 independent experiments
Fig 3: Concentration-dependent effects of AAC inhibitors on cellular metabolic activity in ciPTEC wild type and AAC3-/-. Mature wild-type ciPTEC-OAT1 cells (black), AAC3-/--1 (dark grey) and AAC3-/--2 (light grey) were cultured, matured and exposed to increasing concentrations of AAC inhibitors bongkrekic acid (BKA, A), CD437 (B), carboxyatractyloside (CATR, C), suramin (D) or 0.1% DMSO for 24 h in medium containing 10 mM galactose, followed by measurement of cellular metabolic activity by means of MTT. All results were normalized to DMSO vehicle controls. Statistical analyses: one-way ANOVA corrected for multiple comparison by Dunnett’s post hoc analysis to compare differences between vehicle control and exposed conditions within a cell line: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, indicated in the upper table of each graph) and two-way ANOVA corrected for multiple comparison by Bonferroni’s post hoc analysis to compare overall significant differences between knockout cell line and wild type concentration-dependent responses, as indicated (pAAC3-/--1 or pAAC3-/--2). Mean ± SEM; N = 3 independent experiments
Fig 4: Genetic and chemical AAC3 inhibition reduce mitochondrial respiration. Mature wild-type ciPTEC-OAT1 cells (black and orange/red) were exposed to 0.1% DMSO (black) or 100 µM AAC inhibitors BKA (light orange), CD437 (middle orange), CATR (dark orange) and suramin (red) medium containing 10 mM galactose at 37 °C and 5% (v/v) CO2. Generated AAC3 knockouts (AAC3-/--1, dark grey and AAC3-/--2, light grey) were incubated with 0.1% DMSO in medium containing 10 mM galactose at 37 °C and 5% (v/v) CO2. After 12 h, mitochondrial respiration was evaluated using the Seahorse XF Analyzer. Oxygen consumption rates for all mitochondrial complexes were investigated. Data shown represents basal (A) and maximal respiration (B) and spare capacity (C). Data was corrected for cell count, assessed by fluorescence microscopy after Hoechst staining. Significance was determined by one-way ANOVA with Dunnett’s post hoc analysis, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, mean ± SEM, N = 3 independent experiments, comparing all conditions to wild type (black). Results of basal and maximal respiration and spare capacity shown in panels A–C for wildtype (black) and AAC3-/- (dark and light grey) are also presented in Fig. 2
Fig 5: Stimulation of oxidative respiration by 10 mM galactose does not increase mitochondrial respiration in ciPTEC wildtype and AAC3-/-. The mito stress test assessed key parameters of mitochondrial function by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) upon consecutive injection of oligomycin, FCCP and a combined injection of rotenone and antimycin, using Seahorse XF Analyzer (A and B). Spare capacity was determined by subtracting basal from maximal respiration rates (C). Cellular ATP production was assessed by measuring the ATP rate using the Seahorse XF Analyzer (D). Data was corrected for cell count, assessed by fluorescence microscopy after Hoechst staining. Significance was determined by one-way ANOVA with Dunnett’s post hoc analysis, *p < 0.05, **p < 0.01 and ***p < 0.001, mean ± SEM, N = 3 independent experiments
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