Fig. 1: p16h cells increase with aging in the mouse bladder.
a, Representative scatter plots of Tomato+ (p16h) cells in the bladders of male p16-CreERT2-tdTomato mice at the indicated ages. WT, wild-type. Boxes indicate gating of Tomato+ nonimmune cells. b, Population of Tomato+ (p16h) cells in male mouse bladders at the indicated ages, as determined by fluorescence-activated cell sorting (FACS) (n = 3 for each group). c, Representative scatter plots of Tomato+ (p16h) cells in the bladders of female p16-CreERT2-tdTomato mice at the indicated ages. Boxes indicate gating of Tomato+ nonimmune cells d, Population of Tomato+ (p16h) cells in female mouse bladders at the indicated ages, as determined by FACS (3-month-old mice, n = 3; 12-month-old mice, n = 3; 22- to 25-month-old mice, n = 5). e, Representative images of IHC staining of three layers of the bladder wall in male p16-CreERT2-tdTomato mice at each age. The Tomato protein was stained using an anti-RFP antibody. The areas outlined by dashed lines are the urothelium or lamina propria regions. Scale bar, 100 μm. f, Number of Tomato+ (p16h) cells in the indicated layers of the bladder wall of male mice at each age (n = 3 for each group). g, MFI of FITC signals with or without SPiDER β-gal staining among Tomato+ (p16h) or Tomato− (p16l) cells from young (7- to 12-week-old) male p16-CreERT2-tdTomato mice. The dotted lines indicate each MFI. There is an overlap of the fluorescence spectrum between PE (Tomato) and FITC. Thus, SPiDER β-gal signals were calculated by measuring the ΔMFI of FITC signals with or without SPiDER β-gal. h, ΔMFI of FITC signals among Tomato+ (p16h) or Tomato− (p16l) cells (n = 7). One-way ANOVA with Tukey’s test was performed for b and d and with the Games–Howell test for f. A paired t test was performed for h. Data in b, d, f and h are presented as mean ± s.e.m. All t tests were two-sided.
