Table 1.
Patient data.
Figure 1.
Measurement of growth, ROS and ATP in control and patient fibroblasts.
Fibroblast from control and patient (NDUFS2) were grown in microtiter wells in GLU, GAL or GAL supplemented with AICAR. The amount of control cells, measured by methylene blue (MB) at A620 was compared to viable count by trypan blue at 72h (A). Growth of control and patient cells was measured at 24,48,72h and 144h by MB at A620 (B,C). Growth with AICAR was assessed at 72h (C). ROS measured by DCF at 72h is expressed as relative fluorescent units (RFU) divided by the amount of cells measured by MB at A620 (D). ATP was measured at 72h by luciferin-luciferase is expressed as relative luminescence units (RLU) divided by the amount of cells measured by MB at A620 (E). Values are presented as mean of triplicates +/- standard deviation. *p<0.05.
Figure 2.
The effect of various compounds on growth, ROS and ATP in control and patient fibroblasts.
Fibroblast from three separate controls and seven patients (C20ORF7,NDUFS2,NDUFS4,C6ORF66, FOXRED1 and B17.2L) were grown in microtiter wells in GLU or GAL medium or in GAL medium, supplemented with one of eight various compounds (BEZA,AICAR,OLTI,SBP,RZV,GENI,EGCG,GSI) in GAL for 72hrs. Growth was assessed by MB (A). ROS was measured by DCF and normalized to MB (B). ATP was measured by luciferin-luciferase and normalized to MB (C). Values (A-C) are presented as relative values graphically presented as heatmaps representing the mean of triplicates performed on at least 2 separate occasions. Growth in GAL without additives was set as the value of 1(black). Values < 1 are presented in increasingly green and values >1 in increasingly red. All parameters are summarized in (D) and evaluated as a significantly positive (+), negative (−) or nonsignificant (ns) effect. Increased values for growth and ATP were regarded as positive while negative for increased ROS. Positive only is shaded in red, negative only in green, nonsignificant in dark grey and mixed responses in gray.
Figure 3.
The effect of AICAR on mitochondrial content and Δψ.
Control 1 and an NDUFS2 patient's fibroblasts were grown in GLU or GAL medium and in GAL supplemented with 0.5 mM AICAR (GAL+AICAR) for 72 hrs. Cells were then incubated with TMRE (red) and Mitrotracker green (green) and examined by confocal fluorescent microscopy. A: depicts a representative micrograph TMRE stain in red and MTG stain in green. The graphs represent green intensity per cell (B) and red:green ratio (C), +/− standard deviation (*p<0.05). All micrographs were taken under the same conditions.
Figure 4.
Control 1 (A) and NDUFS2 patient's (B) fibroblasts were grown in GLU or GAL medium and in GAL supplemented with 0.5 mM AICAR (GAL+AICAR) for 72 hrs. Media were changed to unbuffered media and oxygen consumption rates (OCR) were measured by an XF24 instrument. Basal OCR was measured (basal) before the addition of uncoupler and maximal rate (CCCP) after. The results are presented as rates subtracted by non mitochondrial oxygen consumption (in the presence of rotenone and antimycin) and normalized to cell amount measured by methylene blue (MB)at A620, +/− standard deviation. *p<0.05.
Figure 5.
The effect of AICAR on pAMPK and mitotracker red stain in control and patient fibroblasts.
Control and patient fibroblasts were grown on coverslips in GLU or GAL medium and in GAL supplemented with 0.5 mM AICAR (GAL+AICAR) for 72 hrs. Cells were than incubated with mitotracker red (MTR), fixed, stained with anti pAMPK antibodies and visualized by fluorescent (pAMPK) secondary antibodies. The coverslips were examined by confocal fluorescent microscopy (10×40). A: depicts a representative migrograph of pAMPK stain in green and MTR stain in red. The graphs represent green intensity per cell (B) and red intensity per cell (C) +/− standard deviation (*p<0.05). All micrographs were taken under the same conditions.