Apc and p53 interaction in DNA damage and genomic instability in hepatocytes
Runnin Title: Apc and p53 interaction in genomic instability in liver
Valérie Méniel$1, Matthias Megges2, Madeleine A Young1, Alicia Cole3, Owen J Sansom3,
Alan R Clarke1
Supplementary data:
SupFig1: Nuclear -catenin staining showing nearly 100% recombination, cell size, increase
in proliferation in APC and APCP53 and decrease in binucleate cells after Apc loss.
(A) Nuclear -catenin IHC was used as a surrogate marker for APC recombination in APC
and APCP53. (B) More than 80% of Apc is lost at D4 and D6 after induction with naphthoflavone. Same level of recombination in APC and APCP53 (C) Histograms represents
the liver weight in grams per mouse. Apc loss in APC mice leads to hepatomegaly after
induction with -naphthoflavone compared to Wt (N=3, P=0.0404 MW). The additional loss
of p53 does not modify the hepatomegaly phenotype observed in APC (APC vs APCP53, N=3,
P=0.6625 MW). (D) Cell size in m2 was measured in Wt, APC, P53 and APCP53 at D4 and D6
after the cell membrane was stained for -catenin (N>200 cells). (E) Brdu and (F) Ki67
immunostaining in Wt, APC, P53 and APCP53. Arrows indicate positive cells (Scale=10m).
G: Apc loss leads to a decrease in the number of binucleate cells (2 nuclei per cell). The
counting was performed after -catenin IHC where -catenin staining was used to identify
the cell membrane. (N=3 mice/genotype, at least 1000 cells were counted). Bars indicate
standard error. * indicates P<0.05 by MW: Wt, AhCre+Apcfl/fl (APC), AhCre+P53-/-(P53),
AhCre+Apcfl/flp53-/-(APCP53) (N=3 mice for each genotype). (Scale=10m).
SupFig2: Nuclear area in Wt and P53, P21 and P53 staining increased in APC and APCP53
and liver histology .
(A-B): Cumulative plot of nuclei area in Wt vs P53 at D4 (F) and D6 (G) to show the increase
in nuclear area in P53 vs Wt (P=0.01, Kolmogorov–Smirnov, n≥200). C) P53 scoring in APC
versus Wt. Increased % P53 positive cells in APC versus Wt. (D) P21 scoring in APC versus
Wt. Increased % P21 positive cells in APC versus Wt and in APCP53 versus APC, P53 and
Wt.(E)H&E staining showing histology of the liver. Histology of Wt, APC, P53 and APCP53
mice do not reveal any morphological differences between APC and APCP53 livers. Wt,
AhCre+Apcfl/fl (APC), AhCre+P53-/-(P53), AhCre+Apcfl/flp53-/-(APCP53), (Scale=100m), Bars
indicate standard error. * indicates P<0.05 by MW: (N=3 mice for each genotype).
SupFig3: DNA content by Flow cytometry of hepatocytes at D4 and D6 in S phase. We
observed no significant difference in cells in S phase between Wt and APC at both time
point. Wt, AhCre+Apcfl/fl (APC), AhCre+P53-/-(P53), AhCre+Apcfl/flp53-/-(APCP53), Bars indicate
standard error. * indicates P<0.05 by MW: (N=3 mice for each genotype).