Research Paper Volume 5, Issue 1 pp 37—50

Figure 4. Lipofuscin accumulates and co-localizes with Senescence-Associated beta-galactosidase (SA-β-gal) in E2F-1 induced U2OS senescent cells. (A) On the 10^th day of induction with 4-OH-Tamoxifen, cells were positive for SA-β-gal activity (turquoise color); cells also demonstrated the morphological phenotype of senescence (enlarged and flattened) (B) Cells demonstrating the characteristic senescent phenotype show Sudan Black B (SBB) dark blue-black granules (C) Top panels: Lipofuscin's auto-fluorescence at 450-490 nm is represented in green pseudocolor. Bottom panels: blocking of lipofuscin auto-fluorescence (FM, fluorescence microscopy) with SBB staining (BF, bright field microscopy) indicates that SBB stains lipofuscin (D) Concurrent positivity for SA-β-gal activity and SBB staining in the same cell, which is also negative for the proliferative marker Ki67 (E). (F) Addition of 300 nmol/L 4-OH-Tamoxifen (4-OH-Tam) leads to nuclear translocation of E2F1 (indirect immunofluorescence). E2F1-negative nuclei are indicated with white dashed lines. Brown dashed lines: Ki67- negative nuclei, black arrows: SBB granules, NFR: nuclear fast red counterstain.