JCS019372 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. TERT overexpression delays growth arrest under hyperoxia. (A) Frequencies of Ki67-positive nuclei in MRC5 (filled circles) and MRC5-hTERT (open circles) versus time under hyperoxia. For comparison, the value measured in MRC5 grown to senescence under normoxia is also given (filled square). Data are the mean ± s.e.m. from three independent experiments. (B) Frequencies of γH2A.X-positive nuclei (circles) and of Sen-β-Gal-positive cells (triangles) in MRC5 (filled symbols) and MRC5-hTERT (open symbols) versus time under hyperoxia. Data are the mean ± s.e.m. from three independent experiments.

• Supplemental Figure S2 -

  Fig. S2. Set-back of MRC5-hTERT cells to normoxia rescues hyperoxia-induced cell cycle arrest DNA-damage response and telomere shortening. MRC5-hTERT cells were grown under 40% oxygen (filled symbols, straight lines) and set back to normoxia at the indicated times (open symbols, dotted lines). (A) Net cell growth as measured by cumulative PDL. (B) Fractions of Ki67-positive cells. (C) Fractions of γH2A.X-positive cells. (D) Telomere length. Data are the mean ± s.e.m. from at least three parallel measurements per experiment.

• Supplemental Figure S3 -

  Fig. S3. TERT accumulates in mitochondria following treatment with H₂O₂. (A) Purity of nuclear and mitochondrial fractions. 30 µg mitochondrial (M) protein and 20 µg nuclear (N) protein from control or 500 µM H₂O₂-treated MRC5-TERT cells were western blotted and immunostained with antibodies against HDAC2 (nuclear) and COX2 (mitochondrial). (B) Mitochondrial and nuclear fractions as above were western blotted and probed with TERT antibody. (C) TERT chemiluminescence relative to loading control after 500 µM H₂O₂ (in % of untreated control). Data are the mean ± s.e.m. from three experiments.

• Supplemental Figure S4 -

  Fig. S4. TERT overexpression protects mtDNA against nick accumulation. mtDNA damage was measured as fraction of relaxed DNA that allows amplification already after 2-minute heat denaturation. Time under hyperoxia is given in days. Data are the mean ± s.e.m. from triplicate measurements.

• Supplemental Figure S5 -

  Fig. S5. TERT overexpression decreases ROS production in BJ fibroblasts. (A) Mitochondrial superoxide levels are measured by MitoSOX fluorescence in FACS (arbitrary units) in BJ and BJ-TERT fibroblasts. Data are the mean from one experiment. (B) Cellular peroxide levels as measured by DHR123 fluorescence. Data are the mean ± s.e.m. from six experiments. Differences between parental and TERT overexpressing cells (under normoxia and under hyperoxia) are significant with P<0.05 (ANOVA).

• Supplemental Figure S6 -

  Fig. S6. Effect of TERT on expression of genes involved in retrograde response. Expression levels of the indicated genes in MRC5-TERT (TERT), senescent MRC5 (SEN) and young MRC5 (YOU) were measured by semi-quantitative RT-PCR. N, negative control (no RNA); GAPDH, loading control.