Fig. 2: Cycling status versus quiescence of Lgr5^+CBC cells.

a Confocal images showing the crypt-base region of the wild-caught mouse (12-months-old) and wild-caught (12-months-old) NMR small intestine co-stained with Lgr5 RNAscope probe (red), anti-Ki67 antibody (green) and DAPI (blue). Asterisk (*) indicates Lgr5-expressing cells at the crypt base (Lgr5^+CBC) and white arrows mark Lgr5^+CBCKi67⁺ cells. Bar graph showing the mean percentage (±SEM) of Ki67⁺ cells in the total population of Lgr5^+CBC cells per crypt in mouse and NMR small bowel (n = 126 crypts counted from 3 animals/group, P < 0.0001, t-test). b Confocal images showing the crypt base of mouse, human and NMR distal colons co-stained with species-specific Lgr5 or LGR5 RNAscope probe (red), anti-Ki67 or KI67 antibody (green) and DAPI (blue). Asterisks (*) indicate Lgr5^+CBC and white arrows mark Lgr5^+CBCKi67⁺ cells. Bar graph showing the mean percentage (±SEM) of Ki67⁺ cells in total Lgr5^+CBC cells per crypt of NMR and mouse colon (n = 80 crypts counted from 3 animals/group, P = 0.72, t-test) and the mean percentage (±SEM) of KI67⁺LGR5^+CBC cells per crypt in human colon (n = 65 crypts from 4 normal human samples, P < 0.0001, t-test). c Confocal images of mouse and NMR small intestinal crypt base co-stained with species-specific Lgr5 RNAscope probe (red), anti-p27 antibody (green) and DAPI (blue). Asterisks (*) indicate Lgr5^+CBC. All Lgr5^+CBC cells in the mouse and NMR small intestinal crypts were negative for p27. Bar graphs showing the mean percentage (±SEM) of Lgr5^+CBCp27⁺ cells per crypt (n = 51 crypts counted from 3 animals/group). d Confocal images of the crypt base in mouse, human and NMR colon co-stained with species-specific Lgr5 or LGR5 RNAscope probe (red), anti-p27 or P27 antibody (green) and DAPI (blue). Asterisks (*) indicate Lgr5^+CBC or LGR5^+CBC cells. White arrows show LGR5^+CBCP27⁺ cells in the human crypt. White circles mark p27 positive cells in mouse and NMR crypts. Bar graphs showing the mean percentage (±SEM) of p27⁺ or P27⁺ cells in the total population of Lgr5^+CBC or LGR5^+CBC cells per crypt (n = 50 crypts counted from 3 animals/rodent group and n = 30 crypts from 4 human samples, P < 0.0001, t-test). In all cases, Student’s t-tests using two-tailed, unpaired and unequal variance was used. Scale bars are indicated on the images (20 µm).