Fig 4.

SFRP1 induces senescence. (A and B) SFRP1 inhibits cell proliferation. IMR-90 cells were infected with empty lentiviral vector or C-terminally FLAG-tagged SFRP1 lentivirus and were selected with 2 μg/ml puromycin. (A) BrdU incorporation. Four days after infection, the cells were labeled with BrdU for 24 h and stained with antibodies against BrdU and SFRP1. Immunofluorescence microscopy was used to detect the cells displaying no SFRP1 or low SFRP1 expression (Low SFRP1) and those displaying high SFRP1 expression (High SFRP1), and the percentage of BrdU-positive cells was scored. (B) Ki-67 staining. Five days after infection, the cells were stained for Ki-67 and FLAG (SFRP1). *, P < 0.05. (C and D) SFRP1 lentivirus induces senescence phenotypes. IMR-90 cells were infected with empty lentiviral vector or SFRP1 lentivirus and were selected with 2 μg/ml puromycin. Five days after infection, the cells were stained for SA-β-Gal (C) or SAHF (D). *, P < 0.05 compared to vector control. (E) SFRP1 induces senescence-associated secretory phenotype genes. The expression of CXCL1, IL-1α, and IL-8 was examined by RT-PCR 5 days after SFRP1 viral expression of IMR-90 cells. RNA polymerase II (Pol II) served as a loading control. (F) Trypan blue staining. IMR-90 cells expressing SFRP1 (5 days after infection) or Bik (2 days after infection) were assessed for cell death by dye exclusion assays. (G) Caspase 3 cleavage. IMR-90 cells expressing SFRP1 or Bik were assessed for caspase 3 cleavage by immunoblotting with tubulin as a loading control. (H) PARP cleavage. IMR-90 cells expressing SFRP1 or Bik were assessed for PARP cleavage by immunoblotting with nucleolin as loading control. (I) Recombinant SFRP1 induces senescence in IMR-90 cells. IMR-90 cells were treated with the indicated concentration of recombinant SFRP1 for 4 days and were stained for SA-β-Gal. *, P < 0.05 compared to untreated control. (J) Nonsenescent IMR-90 cells acquire a senescence phenotype upon coculture with senescent cells. Nonsenescent IMR-90 cells were infected with GFP-expressing lentivirus. These GFP-labeled IMR-90 cells were cocultured with either senescent IMR-90 cells (induced to senesce by etoposide treatment [left] or by SFRP1 lentiviral expression [right]), or nonsenescent IMR-90 cells (untreated or vector infected) for 4 days. Where indicated, the cells were treated with 1 μg/ml of control IgG or anti-SFRP1 antibody beginning 24 h after initiation of coculture. The GFP-positive cells were scored for the development of senescence-associated heterochromatic foci (SAHF). *, P < 0.05. Note that the GFP-positive cells in coculture were not positive for the DNA damage marker γH2AX, indicating that the spreading of senescence is not due to residual etoposide or SFRP1-induced DNA damage.