Figure 3.

Identification and quantification of senescent cells in vivo. (A–F) MEF tumor cells expressing tetracycline transactivator protein tTA (‘tet‐off’), H‐ras^V12, GFP, and TRE‐shp53 were injected subcutaneously into nude mice. Once tumors appeared, mice were treated with (+DOX) or without (−DOX) doxycycline. Tumors were extracted, dissociated, stained for SA‐β‐gal and CD45 (yellow), and analyzed by ImageStreamX. Bar, 10 μm. (A) Representative images of GFP‐positive tumor cells treated with or without DOX. (B) BF mean pixel intensity distribution of +DOX and −DOX tumor cells. (C) Quantification of positive tumor cells, as gated in (B). (D) Representative images of immune cells and tumor cells extracted from DOX‐treated mice. (E) BF mean pixel intensity distribution of immune cells extracted from DOX‐treated and untreated mice. (F) Quantification of positive immune cells, as gated in (E). (A–F) Data (n = 5 mice) were replicated in two independent experiments. Values are mean ± SEM ***P < 0.001 (Student's t‐test). NF = Normalized frequencies.(G–I) Lungs were extracted and dissociated from bleomycin‐treated and untreated mice. Cells were stained for SA‐β‐gal, CD45 (yellow), DAPI (blue), and Pan‐cytokeratin (PCK, red) and analyzed by ImageStreamX. (G) Representative images of the identified cells are shown. Bar, 10 μm. (H) Quantification of positive epithelial cells. (I) Quantification of positive immune cells. Data (n = 4 mice) were replicated in two independent experiments. Values are means ± SEM, ***P < 0.001 (Student's t‐test). (J) Quantification of Ki67‐ and BrdU‐negative cells within SA‐β‐gal‐positive and SA‐β‐gal‐negative lungs cells of bleomycin‐treated mice. n = 3, ***P < 0.001, **P < 0.01 (two‐way ANOVA).