Figure 6. Loss of RASSF1A results in altered intestinal homeostasis.

(A) Gut permeability of the indicated animals was determined by FITC-dextran fluorimetry on day 9 post-DSS addition. P values (all + DSS): wild-type vs Rassf1a^−/− = 0.007; wild-type vs Rassf5a^−/− = 0.4; Rassf1a^−/− vs Rassf5a^−/− = 0.005, wild-type vs Rassf1a^IEC-KO = 0.0001, n = 6 – 10 per group. (B) Right panel, bacterial translocation to blood and mesenteric lymph nodes as indicated (liver and spleen also revealed some translocation). Left panel, histogram plot of the percentage of these four areas colonized with bacteria. (C) Analysis for PARP cleavage was carried out as indicated on day 9 post-DSS treatment. “*” p value  = 0.0028 and “**”  = 0.0295, n = 5 – 7 for all genotypes and treatments. P value wild-type (+DSS) vs Rassf5a^−/− (+DSS)  = 0.989. (D) TUNEL positive staining (bright dots) was carried out as a late marker of cell death. These pictures are a representative of three independent histological sections from each genotype and treatment. The mean number of cells/crypt shown was counted 6 times. P value wild-type (+DSS) vs Rassf1a^−/− or Rassf1a^IEC-KO (+DSS)  = 0.002 and 0.04 respectively. (E) Colon lysates (top panel) or nuclear fraction of isolated crypt cells (bottom panel) from the indicated genotypes were used to detect total or phosphoserine (S)127 YAP, total YAP and p73 in the nuclear fractions. For the top panel, the +DSS lanes are colon lysates from different DSS-treated animals. All treatments and genotypes had similar levels of total p73 following total lysate analysis by immunoblotting (data not shown). (F) Detection of total or pY-YAP as indicated. Purity of nuclear and cytoplasmic fractions are shown in Figure S6E. For (C – D) all untreated results for Rassf1a^−/−, Rassf5a^−/− and Rassf1a^IEC-KO (untreated) were similar to wild type (untreated).