JCS059246 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Reduced expression of Drp1 and Fis1 in western blotting. Total cell lysates of young and old HUVEC from three different isolations (A, B and C) were separated by SDS-PAGE and analyzed by Western blot with anti-Drp1, anti-Fis1 and anti-actin antibody for normalization. Quantification is shown in Fig. 1C.

• Supplemental Figure S2 -

  Fig. S2. mRNA levels of fission and fusion factor in young and old HUVEC. Relative mRNA levels of SLP2, Mfn1, Mfn2, Opa1 and MTP18 in young and old HUVEC from four to six different isolations were determined by semiquantitative RT-PCR and again by qPCR. The mRNA expression levels of young cells were set as 100% respectively. The SLP2 transcript was significantly downregulated, p<0.001, n=4. The mRNA expression levels of the other depicted fission and fusion factors were not significantly altered.

• Supplemental Figure S3 -

  Fig. S3. Growth curves of control and irradiated HUVEC. Young HUVEC were either stained with 25 nM MTR and not irradiated (control S) or irradiated without staining (control I) or stained with MTR and irradiated (0.3 J/cm²) and cultivated till senescence. Stained and irradiated cells reached senescence in the same time frame as the controls.

• Supplemental Figure S4 -

  Fig. S4. Irradiation of stained mitochondria did not induce cytochrome c release. Young cells were either stained with MTR and not irradiated (control S), or irradiated without MTR staining (control I) or stained with MTR and irradiated (0.3 J/cm²). 8 h after irradiation an immunostaining against cytochrome c was performed. Both controls as well as stained and irradiated cells show a colocalisation of MTR and cytochrome c despite the fragmentation of mitochondria in the MTR stained and irradiated sample.

• Supplemental Figure S5 -

  Fig. S5. Irradiation of stained mitochondria did not induce apoptosis in young cells. Young HUVEC were either stained with MTR and non-irradiated (control S), or irradiated without staining (control I), or stained with MTR and irradiated (0.3 J/cm²). The total and apoptotic nuclei were counted at the indicated time points (at least 100 cells/sample). For both controls, only the average over the whole time is represented. No increase of apoptosis after irradiation was observed; (n=3).

• Supplemental Figure S6 -

  Fig. S6. Irradiation of stained mitochondria did not induce apoptosis in old cells. Senescent HUVEC were either stained with 40 nM MTR and not irradiated (control S) or irradiated without staining (control I) or stained with MTR and irradiated (0.3 J/cm²). For both controls only the average over the whole time is represented. The apoptotic rate was about 10% higher in old cells compared to young HUVEC, but irradiation of stained cells induced no significant changes in comparison to the controls; at least 100 cells/sample; n=3.