Establishment of Flow-FISH reference curves for telomere length

A. The age-related reference interval and centiles were calculated after telomere length measurement by Flow-FISH in lymphocytes obtained from 491 healthy donors. The P50 curve is shown in dark blue. Other percentile curves are shown as indicated in the legend. B. Same as A in granulocytes. C. TL in lymphocytes (left) or granulocytes (right) of eight selected donors. D. Mitochondrial subhaplogroups of the eight selected donors with an overview of the variants showing both a change in the amino acid sequence and an overall frequency of less than 10% in Mitomap database in the mtDNA coding regions of the donors. The variant frequency in the subhaplogroup of each donor is indicated in the last column.

Mitochondrial genomes modulate telomere length and integrity of ρ0 recipient cells.

A. Left: Representative Southern blot analysis (TRF) of telomeres in 143B, 143B ρ0 recipient cells and in one cybrid clone from each donor at the first PDs post-selection. Right: Quantification of TRF on four independent cybrid clones for each. Mean ± SEM. One-way ANOVA. ns: p>0.05. B. Left: TRAP assay products for the indicated cell lines. Right: ddTRAP quantification of telomerase activity in the indicated cell lines. Copy number concentrations were normalized to the first replicate of 143B ρ0 cells (repl 1). Mean ± SEM. One-way ANOVA. C. qRT-PCR analysis of hTR and hTERT transcripts. Values were normalized first to ACTB mRNA levels and then to 143B ρ0 ratio. D. Left: Representative pictures of telomere dysfunction-induced foci (TIF) detection by co-localization of telomeres (red FISH probe) with 53BP1 DNA damage marker (green). White arrowheads indicate TIF. Blue: DAPI. Scale bar: 5 μm. Right: TIF quantification in clone #1 of all cybrids (first PDs post-selection) and in 143B ρ0 cells. n=47-63 nuclei. Mean ± SEM. Kruskal-Wallis test. ns: p>0.05. E. Visualisation of telomere fusions (white arrowheads) in Cyb1 cells at PD7. 143B ρ0 and Cyb6 cells are shown as controls. Telomeres are detected by FISH (red) and DAPI stains DNA (blue). Scale bar: 5 μm.

Telomere shortening in cybrids from donor with low complex I activity is counteracted by antioxidant and NAD+ precursor.

A. Complex I (CI) activity in 143B and in the cybrids. Values were normalized to the activity of complex II (CII) which is entirely nuclear-encoded. n measurements from 2 biological replicates on clone #1 from all: at PD 4-7 or PD 16-24 post-selection. Mean ± SEM. Kruskal-Wallis test. ns: p>0.05. B. Correlation between TL in the cybrids at early PDs and average ratios of CI/CII activity. Color code as in A. C. Expression of NAMPT and NAPRT1 genes from the NAD+ salvage pathway in cybrids (PD≥5 post-selection) from donors with either low (Cyb1 to 2) or normal (Cyb3 to 7) CI activity. Values were normalized to ACTB and to the ratios measured in 143B cells. Color code as in A. n=3 biological replicates. Mean ± SEM. Two-tailed Student’s t tests. D. Representative TRF analysis of TL in cybrids obtained by fusion between donor #2’s platelets and 143B ρ0 recipient cells in the presence of the indicated treatments or in the absence of added drug (−). 5 mM N-acetyl-L-cysteine (NAC), 5 mM N-acetyl-L-alanine (NAA, control) and/or 3 mM nicotinamide riboside (NR) were added at the time of the fusion and kept until clone isolation. E. TL was measured in the following numbers of independent clones: 5 (NAA), 7 (NAC), 8 (NAC + NR or NAA + NR) and the percentage of clones with average TL>2.5 kb was calculated. F. Representative images and quantification of telomeric parylation in Cyb2 cells comparing cells that did or did not exhibit telomere shortening during the initial PDs following cybridisation (from panel D-E). Parylation was detected by IF (green) in the presence or PARG inhibitor; telomeres were detected by FISH (red) and DNA was stained with DAPI (blue). Scale bar: 5 μm. n=94-116 nuclei were counted for each clone. Mean ± SEM. Two-tailed Mann-Whitney test.

Mitochondrial superoxide production in cybrids inversely correlates with TL in lymphocytes.

A. Lack of correlation between lymphocyte TL (in kb, adjusted to 50 yo) and average CI activity measured in cybrids from the corresponding donors and normalized to CII activity (CI/CII). B. Mitochondrial superoxide production measured by electron paramagnetic resonance and corrected for background using PEG-SOD2 in clone #1 from all cybrids. n measurements from 2 biological replicates with 3 technical replicates. Mean ± SEM. Kruskal-Wallis test. ns: p>0.05. C. Correlation between lymphocyte TL (in kb, adjusted to 50 yo) and average mitochondrial superoxide production measured in cybrids from the corresponding donors. D. Complex IV activity in 143B cells and in the cybrids. Values were normalized to CII activity. n measurements per cell line from 2 biological replicates. One-way ANOVA. ns: p>0.05. E. TL of donor #7 (H1b1e), donor #1 (T2b4a), donor #6 (K1a4a1) and their relatives is shown as indicated in the legend. Orange triangles show TL in unrelated K1a11b and K1a4a1a2b donors. F. Frequency of donors with the ATP6 A177T variant among n selected donors with lymphocyte TL in the indicated ranges (<P50, between P50 and <P90 and ≥P90). Chi-square test.