Cell cycle analysis of nonhealer and healer ear-derived cells. (A) Control cells (NIH 3T3); (B–D) dermal cells derived from the ear pinnae of nonhealer B6, SM/J, and RI line 33, respectively; (E–H) healer MRL/MpJ, congenic, LG/J, and RI line 6, respectively, were seeded at the same number and grown for 3 days. Cells were harvested and labeled with propidium iodide and analyzed by flow cytometry analysis for stage of cell cycle, G0/G1, S, G2/M. The y axis shows the number of cells counted and the x axis shows an increasing amount of propidium iodide incorporation/cell (Left to Right).

p53 expression in cells and in both normal and injured tissue. Cells from healer mice (MRL, cong) and nonhealer mice (B6) were cultured on coverslips and then stained with DO1 anti-p53 antibody and anti-mouse Ig + DAPI. (A) p53 nuclear staining (anti-p53 is green and DAPI is blue) in an MRL cell (Left) and a B6 cell (Right). (B) The percent p53-positive cells in MRL, cong, and B6 cultured dermal cells. (C) The percentage of p53-positive cells at each stage of the cell cycle is indicated for the MRL cell line. Cells were stained with Vybrant Orange, separated by flow cytometry into three cell cycle stages, G0/G1, S, and G2/M. These cells were then put on slides and stained with antibody to p53. (D–K) shows histological sections of normal ear and small intestine from nonhealer (B6) (D, F, H, and J) and healer (MRL) mice (E, G, I, and K), which were stained with anti-p53 antibody + DAB. Western blots from normal B6, MRL, and congenic ear tissue along with Coomassie-stained gels as tissue loading controls are shown (L). Samples were run on a 14% SDS/PAGE gel and stained with anti-p53 antibody. Tissues from B6 and MRL injured ears (M) from Left to Right (B6, MRL day 0, 5, and 10). Above the p53 gel is a Coomassie blue-stained gel as a tissue loading control run per lane.

Detection of γH2AX and TopBP1 foci in cells and normal tissue. Dermal cells from healer mice (MRL, Cong) and nonhealer mice (B6) were cultured on coverslips and then stained with antiphospho-histone H2AX). White bars are normal cells, black bars are irradiated cells (1 Gy). (A) The number of foci/cell by counting 20 nuclei/cell line at 60× is presented as three histograms (Aa) with MRL (Top); Congenic (Middle), and B6 (Bottom). (Ab) Percentage of γH2AX-positive cells determined by counting between 100–200 cells/treatment. (Ac) A representative γH2AX-stained MRL cultured dermal cell nucleus with foci. (Ba–d) Tissue sections from ear and small intestine of normal B6 and MRL mice treated with γH2AX antibody and DAB stains hair follicles, dermal and basal epidermal cells in the ear, and villous epidermal cells in the small intestine. (Ca) Western analysis of ear-derived dermal cells and (Cb) ear tissue is shown using chromatin-enriched tissue run on a 14% SDS/PAGE gel. (Ca) Loading controls for cells (lanes 1, 2) stained with Coomassie blue; γH2AX bands (lanes 3, 4) seen at approximately 15 kDa. (Cb) Loading controls for tissue stained with Coomassie blue (lanes 1–3); γH2AX bands (lanes 4–6). (D a and b) A similar analysis of TopBP1 staining of nuclear foci. (Dc) TopBP1 foci are seen in an MRL dermal cell nucleus.

Functional analysis of DNA damage and repair. (Aa–f) Ear dermal cells from nonhealer B6 and healer MRL mice were tested for DNA damage using the comet assay under alkaline conditions. Comets can be seen in MRL cells at 10× (Ad) and 60× (Ae) magnification but not in B6 cells (A a and b, respectively). (A c and f) After 10 Gy γ-irradiation, all B6 and MRL cells display comet formation. (B) Analysis of Rad 51. (Ba) Rad51 foci in an MRL healer cell; (Bb) percent Rad51-positive cells in MRL, Cong, and B6 cells. (Ca) Western analysis of caspase protein on a 12% SDS/PAGE gel of B6 and MRL tissue samples from small intestine (two samples each) with expression only in MRL. (C b–d) Histological sections from nonhealer (B6) and healer (MRL) ears stained with antibody to the active form of caspase 3. (D a–d). Sections from small intestine were examined by TUNEL at 10× (D a and b) and 60× magnification (D c and d). (E) Western analysis of p21 expression in a human cell line (HCT116), in MRL healer cells, and B6 nonhealer cells in the absence and presence of ionizing irradiation (1 Gy).

P21 knockout mouse ear hole closure. (A) Ear pinnae of p21 KO (CDKN1A^−/−) and WT (B6129F2) control female mice were ear punched and the mean hole diameters ± SD measured. Ear hole closure of KO (n = 10 × two ears) and WT mice (n = 9 × two ears) is compared to B6 and MRL hole diameters and shown weekly over a 1 month period. (B and C) Photographs of p21^+/+ ear holes (day 5 and 35 postinjury, respectively) and (D and E) photographs of p21^−/− ear holes (day 5 and 35 postinjury, respectively) can be seen. (Scale bar: 1mm.) (F–H) Histological analysis shows p21^−/− ear hole tissue on day 35. (F) Ear tissue extending from the cut cartilage (4×). (G) Normal blastema-like tissue (40×) and (H) condensations extending from the cut cartilage can be seen (arrow: 40×). (I–N) Analysis of cells from the ear pinnae of p21^+/+ mice and p21^−/− mice for (I and J) γH2AX staining (DAPI = blue; anti-γH2AX = red), (K and L) comets. (M) Cells were counted for both γH2AX staining and comets and percent positive cells + SD determined. (N) Cell cycle analysis (using propidium iodide) was carried out with the p21^−/− cells in red and p21^+/+ cells in blue. (O) Model describing the links between cell cycle checkpoint control, proliferation, and the tissue regeneration phenotype. After tissue injury such as an ear hole punch, hyperproliferation ensues. In WT or p21-proficient cells, a G1 checkpoint is enacted leading to scar formation. In p21-defective cells such as those derived from the MRL strain and p21^−/− mice, rounds of unscheduled DNA synthesis can occur, leading to DNA damage and an abundance of DNA damage markers. A G2 checkpoint can be enacted, leading to enhanced tissue regeneration.