JCS034876 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Characteristics of cell senescence in cultures of hMSCs using Heyflick senescence-associated factors. (A)Immunofluorescence of hMSCs at passage 6 and 10 show p16(INK4a) (green) and lamin A (red). Dense spots with the DAPI counter staining indicate DNA foci. Scale bars: 20 µm. (B) Combination of SA-β-galactosidase activity with immunofluorescence. Cells at passage 9 were stained for SA-β-galactosidase activity, followed by immunofluorescence with lamin A antibodies (red). DNA was counterstained with DAPI. SA-β-gal staining appears as dark spots, and the nucleus as light gray, after image conversion to grayscale. An overlay of the SA-β-gal staining and lamin A immunofluorescence is shown in the merged image. Scale bars: 20 µm. γ-H2AX foci are formed in senescent hMSCs. Cells at passage 7 were transduced with the lamin B-EGFP lentiviral vector followed immunolabeling with γ-H2AX antibodies. Shown are the nuclei of normal and senescent cells, as determined by the lamina structure. Scale bars: 20 µm.

• Supplemental Figure S2 -

  Fig. S2. Characterization of senescence in the 35-y and 81-y cells. Western blot analyses of p16(INK4a), hTERT and p53 expression in 35-y and 81-y cells at passage 7. Ponceau S shows loading control. (B) SA-β-gal activity and lamin A immunohistochemistry in 35-y and 81-y cells. SA-β-gal activity assay was carried out on cells in passage 6 followed by immunocytochemistry with R-anti-lamin A antibodies, which was labeled with anti-R-Cy5 secondary antibody. DNA was counterstained with DAPI. When combining UV and white light the SA-β-gal staining appears as dark spots, and the nucleus as light blue. The image was converted to grayscale to enhance contrast. Scale bars: 40 µm. Changes in the organization of PML-NBs in 35-y and hMSCs. Cells at passage 3 were transduced with lamin A-DsRed lentiviral vector. PML-NBs was visualized with the corresponded antibodies bound to anti-M-Alexa Fluor 488 (green). Shown are maximum projections of confocal images taken from representative 81-y cells at passage 4 and 7, or 35-y cells at passage 4 and 12. Significantly, the large structures of PML-NBs do not colocalize with lamin A-DsRed.