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. 2003 Jan;23(1):389–401. doi: 10.1128/MCB.23.1.389-401.2003

FIG. 6.

FIG. 6.

FIG. 6.

High-p16-expressing cells in presenescent WI-38 cultures and effect of Bmi-1 overexpression. (A) Presenescent cells at PD 24 (Presen) and senescent (Sen) cells were immunostained for p16 as described in Materials and Methods and photographed (magnification, ×100) at the same exposure for each set. Nuclei were visualized by DAPI staining. HeLa and H1299 cells served as positive and negative controls for p16 expression, respectively. (B) Presenescent WI-38 cells at PD 24 were infected with control (B0) or Bmi-1-expressing retroviruses, stained for p16 and DAPI after selection and photographed (magnification, ×200). (C) Presenescent WI-38 cells at PD 24 were immunostained for p16 and Ki67 as described in Materials and Methods and photographed (magnifications, ×300 [top] and ×400 [bottom]). The arrows show examples of p16-positive cells that are Ki67 negative. (D) Presenescent cells at PD 29 were stained for SA-β-Gal activity and subsequently immunostained for p16 as described in Materials and Methods. SA-β-Gal staining was photographed under phase-contrast and bright field (SA-β-Gal) microscopy. HeLa cells served as a positive control for p16 staining. Magnification, ×200. (E) Presenescent cells (PD 24, Presen) were infected with retroviruses carrying no insert (B0) or Bmi-1 (B-Bmi-1), selected, and passaged until senescence (Sen; %LN < 10%). Cells were further cultured for 2 months (B0-L and B-Bmi-1-L; %LN < 1%). Cell lysates were analyzed by Western blotting for Bmi-1, p16, and α-tubulin (control) proteins.