Figure 1.
TFAM mRNA silencing induces mtDNA depletion 72 h post transfection.
MIN6 cells were transfected with TFAM-193, TFAM-429 or Scrambled siRNA probes, or with no siRNA (Shocked). TFAM mRNA expression was quantified relative to reference gene B2M by real-time PCR at 48 h (A) and 72 h (B) post transfection. mtDNA depletion was also measured by real-time PCR, using mitochondrial encoded ND5 relative to nuclear encoded GAPDH at 48 h (C) and 72 h (D) post transfection. All results normalised to Scrambled negative control. Experiment repeated once (C), twice (A) or 4 times (B, D) in triplicate. Data presented are means ± SEM (SD in (C)). * p<0.05, *** p<0.001.B2M, β2 Microglobulin; GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase; ND5, NADH Dehydrogenase 5; TFAM, Mitochondrial Transcription Factor A.
Figure 2.
The effect of partial mtDNA depletion on mitochondrial gene transcription and protein translation.
Mitochondrial DNA was depleted following TFAM silencing and COX1 mRNA expression was quantified relative to reference gene B2M 72 h post transfection (A). Protein was extracted 72 h post transfection and analysed by western blotting, probing for COX1, SDH70, and β-Actin proteins. Protein bands were quantified by densitometry, and optical density readings used to calculate the ratio of COX1 (B) and SDH70 (C) mitochondrial proteins relative to β-Actin loading control. A representative blot is shown in (D). Data in (A) are normalised to Scrambled control cells. Both experiments repeated 3 times, with each experimental repeat performed in triplicate (A) or duplicate (B, C). Data presented are means ± SEM. * p<0.05, ** p<0.01, *** p<0.001. COX1, Cytochrome c Oxidase 1; SDH70, Succinate Dehydrogenase 70 kDa subunit.
Figure 3.
The effect of partial mtDNA depletion on mitochondrial function.
Cells were harvested 72 h post transfection and oxygen consumption rate (OCR) was measured using the Seahorse XF24 Analyzer. OCR in TFAM-429 cells (n = 8) was severely impaired compared to that of Scrambled (n = 8) and Shocked (n = 8) control cells (A). Mitochondrial activity was measured following injection of oligomycin, an inhibitor of Complex V (ATP Synthase), followed by two sequential injections of FCCP to uncouple respiration and induce maximal respiration, and finally antimycin, an inhibitor of Complex III (Ubiquinol-Cytochrome c Reductase) preventing electron transfer and subsequently abolishing the proton gradient required for ATP synthesis. Basal and maximal respiratory capacity (B) and ATP synthesis by oxidative phosphorylation (OXPHOS) (C) were calculated as described previously [30]–[32]. Data were normalised to protein concentration and are presented means ± SEM. * p<0.5, ** p<0.001, *** p<0.0001.
Figure 4.
The effect of partial mtDNA depletion on glucose-stimulated insulin secretion.
Seventy two hours post transfection cells were stimulated with basal (3 mM) or high (25 mM) glucose concentrations. Insulin secretion (A) and insulin content (B) were determined by insulin ELISA and normalised to protein content. Data normalised to 3 mM glucose stimulated Scrambled control cells. mtDNA levels (C) and Ins1 insulin gene expression (D) were quantified and normalised to the Scrambled control. Data shown are from 9 (A) or 3 (B, C, D) separate experiments, each performed in triplicate. Data presented are means ± SEM. ** p≤0.01, *** p<0.001.
Figure 5.
The effect of glibenclamide on insulin secretion following TFAM silencing-induced mtDNA depletion.
Seventy two hours post transfection cells were stimulated with 3 mM or 25 mM glucose, supplemented with or without 0.1 µM glibenclamide. Insulin secretion was determined by insulin ELISA and normalised to whole cell protein content. Data shown are from 4 separated experiments performed in triplicate, and are normalised to Scrambled negative control cells stimulated with 3 mM glucose without glibenclamide. Data presented are means ± SEM. * p<0.05, ** p<0.01.