Figure 1.
Isolation of SASP-boosted therapy-induced senescent cells
(A-C) UPK10 cells were treated with 10 μM cisplatin for three days. After three days of release, cells were stained for SA-β-gal activity (A) and percentage of SA-β-gal positive cells were quantified (B). Expression of the indicated proteins was also examined by immunoblot in the indicated cells (C).
(D) UPK10 cells were treated with 10μM cisplatin, 10μM irinotecan, or a combination for three days and released for three days. SA-β-gal positive cells were quantified using SPiDER SA-β-gal assay by flow cytometry.
(E and F) UPK10 cells were treated with a combination of 10μM cisplatin and 10μM irinotecan for three days and released for three days. Senescent and non-senescent cells were sorted using gating strategies indicated in (E). Phase contrast images of sorted non-senescent and senescent UPK10 cells after replating were shown (F).
(G) Sorted senescent and non-senescent cells from cisplatin and irinotecan treated UPK10 cells at the indicated time points post sorting (24 hrs or 3 weeks) were labeled with BrdU for 24 hrs and BrdU incorporation was examined by immunofluorescence staining and quantified.
(H) 1 X 106 sorted senescent and non-senescent cisplatin and irinotecan treated UPK10 cells (n=3 mice per group) were orthotopically transplanted into mouse bursa that covers mouse ovary. Shown are images of ovaries with tumor formed by non-senescent cells in one month and those without evidence of tumor formation by sorted senescent cells after two and half months.
Data represent mean ± SEM of 3 biologically independent experiments. Scale bar = 100 μm in 1A and 1F, and = 20 μm in 1G. p values were calculated using a two-tailed t test. See also Figure S1.