Fig 6.

Wnt inhibition results in senescence. (A) SFRP1 reduces soluble β-catenin levels in IMR-90 cells. IMR-90 cells were infected with vector, SFRP1, or Wnt3 lentivirus or treated with 20 μM etoposide. Five days later, soluble β-catenin levels were analyzed by immunoblotting using 5 μg of each soluble fraction. (B) SFRP1 knockdown abrogates etoposide-induced reduction of soluble β-catenin levels. IMR-90 cells were infected with lentiviruses expressing shRNA against luciferase (shLuc) or shRNA against SFRP1 (shSFRP1-3). The cells were treated with 20 μM etoposide, and 5 days later, soluble β-catenin levels were analyzed by immunoblotting using 5 μg of each soluble fraction. (C) SFRP1 silences Wnt-dependent transcription in IMR-90 cells. IMR-90 cells were cotransfected with Super TopFlash reporter, cytomegalovirus (CMV)-β-Gal, and empty vector, SFRP1, or Wnt3 where indicated, and the luciferase activity was determined 48 h after transfection. Transfection efficiencies were normalized using CMV-β-Gal activity. (D) Etoposide treatment represses TopFlash reporter activity. IMR-90 cells were treated with 20 μM etoposide or left untreated for 24 h prior to transfection. The cells were transfected with Super TopFlash reporter and CMV-β-Gal for 4 h, and 1 μg/ml anti-SFRP1 antibody (H-90) or IgG was added after removal of the transfection mixture. The luciferase activity was determined 48 h after transfection. Transfection efficiencies were normalized using CMV-β-Gal activity. *, P < 0.05. (E) Wnt3 counteracts SFRP1-induced senescence. IMR-90 cells were infected with the indicated lentiviruses, and 5 days after infection, the cells were stained for SA-β-Gal. *, P < 0.05. (F) Wnt3 counteracts SFRP1-induced reduction of soluble β-catenin levels. Soluble β-catenin levels were analyzed by immunoblotting. (G) Wnt3 counteracts SFRP1-induced repression of TopFlash reporter activity. IMR-90 cells were transfected with Super TopFlash reporter and CMV-β-Gal in conjunction with SFRP1 or Wnt3 as indicated. *, P < 0.05. (H) LiCl treatment suppresses SFRP1-induced senescence. IMR-90 cells were infected with vector or SFRP1 lentivirus, and 3 days after infection, the cells were treated with 20 mM LiCl for 48 h and stained for SA-β-Gal. *, P < 0.05. (I) Wnt3 counteracts etoposide-induced senescence. IMR-90 cells were infected with Wnt3 or empty vector lentivirus. The cells were treated with 20 μM etoposide or left untreated, and the SA-β-Gal activity was analyzed 6 days after etoposide treatment. *, P < 0.05. (J) Wnt3 counteracts oxidative stress-induced senescence. IMR-90 cells were treated with 500 μM H₂O₂ for 2 h. The subsequent day, the cells were infected with lentiviruses expressing Wnt3 or empty vector. On day 6 after H₂O₂ treatment, the cells were analyzed for SA-β-Gal. *, P < 0.05. (K) Pharmacological inhibition of Wnt signaling results in senescence. IMR-90 cells were treated with dimethyl sulfoxide (DMSO) control or 1 μM pyrvinium pamoate for 4 days and were stained for SA-β-Gal. (L) shRNA knockdown of β-catenin. IMR-90 cells were infected with lentiviruses expressing scrambled shRNA (sh scr), shRNA against β-catenin-1 (sh β-cat#1), or shRNA against β-catenin-2 (sh β-cat#2) (sh β-cat 2) and 5 days after infection, β-catenin protein levels were analyzed by immunoblotting. Nucleolin served as a loading control. (M) β-Catenin knockdown results in senescence. IMR-90 cells were infected with the indicated lentiviruses and at 5 days postinfection stained for SA-β-Gal. (N) All five SFRP family members induce senescence. IMR-90 cells were infected with lentiviruses expressing the indicated SFRP family members. At 5 days postinfection, the cells were stained for SA-β-Gal. *, P < 0.05 compared to vector control. (O) DKK1 induces senescence. IMR-90 cells were infected with vector or DKK1 lentivirus. At 5 days postinfection, the cells were stained for SA-β-Gal. *, P < 0.05 compared to vector control.