Figure 1. Impaired accumulation of senescent cells in Ccn1^dm/dm mice during cutaneous wound healing.

Excisional cutaneous wounds were created using a 6 mm biopsy punch in Ccn1^WT/WT (n=5) and Ccn1^dm/dm mice (n=5). (a) Time course of Ccn1 expression and overlapping phases of healing events. (b) Wound closure rate was monitored by measuring wound diameters on indicated days post-wounding. (c) The numbers of Ki-67-positive cells were counted as a percentage of Hematoxylin-positive nuclei in 5 randomly selected high-powered fields in frozen sections of granulation tissues, and (d) the numbers of apoptotic cells were counted after TUNEL staining. (e) Granulation tissues 7 and 9 days post-wounding were stained for SA-β-gal activity, and counterstained with Eosin. Inserts show the entire wound sections and the boxes indicate the enlarged areas. SA-β-gal positive cells were prominent in WT wounds but virtually undetectable in Ccn1^dm/dm wounds. Similar results were observed in another independently-derived Ccn1^dm/dm knockin mouse line. Scale bar = 50 μm. (f) Granulation tissues 9 days post-wounding were double-stained for p16^INK4a (red) and α-SMA (green) by immunofluorescence, and counterstained with DAPI (blue). (g) Cells isolated from 7- and 9-day wounds were plated in 60 mm dishes and stained for SA-β-gal activity 2 days later (n=3).