Fig. 2: PP2A loss or inhibition is associated with MSI status.
a–g Lgr5-EGFP-CreERT2; Ppp2r1aflox/flox intestinal organoids were treated with 5 μg/ml DMBA, 50 μg/ml MNU, or 10 μg/ml PhIP in combination with or without tamoxifen (TAM) for 50 days. a Total numbers of somatic mutation events calculated from three biologically independent samples in each group display a very high mutational load. Data are denoted as mean ± s.e.m. b Mutational spectra of all base substitutions in organoid cultures treated with the indicated carcinogen and TAM. Similar mutation signatures were observed. c Heat map showing the cosine similarity scores for each indicated sample and COSMIC signature. The samples have been clustered according to the similarity score with each signature. The signatures have been ordered according to their similarity, such that very similar signatures cluster together. d The MSI status was evaluated by comparing mononucleotide repeats in each indicated sample. The mononucleotide regions mBAT-26, mBAT-37, mBAT-24, and L24372 were used to evaluate microsatellite instability. Mutant alleles are indicated with specific base pair as number(s) in each sample trace. Shifting or loss of heterozygosity (LOH) comparing to Control are marked with colour corresponding to each sample respectively. e Heat map of RNA-seq analysis of genes associated with DNA mismatch repair. f Western blotting and g immunohistochemical studies of MLH1 levels in organoid cultures treated with the indicated conditions. Blots are representative of two biological independent samples for each group. Images are representative of three biological independent samples for each group. Bar = 25 μm. h Ppp2r1a-loss colon tumours were induced in Lgr5-EGFP-CreERT2; Ppp2r1aflox/flox by treatment with DMBA, MNU or PhIP, and TAM for 36 days. Control colon tissues (Control) and Ppp2r1a-loss colon tumours were harvested for MLH1 level analysis by immunohistochemistry. Arrows indicate positive signals. Images are representative of three biological independent samples for each group. Bar = 25 μm. i Analysis of mRNA level expression were generated by the software from Gepia (http://gepia.cancer-pku.cn) using the data from TCGA and presented as mean ± s.e.m., revealing higher CIP2A (n = 372, P = 0.0096) and SET (n = 372, P = 0.05) and lower PPP2R1A (n = 372, P = 0.1576) levels in MSI compared to MSS. P value was determined by two-sided unpaired t test (CIP2A, SET) and Mann–Whitney U-test (PPP2R1A). j Human tissue array containing MSI and MSS colorectal tumours was assayed for CIP2A (n = 152, P = 0.0043), SET (n = 150, P = 0.0077) and PPP2R1A (n = 129, P = 0.64) levels by immunohistochemistry. (Left) Representative images are shown. Arrows indicate positive signals. Bar = 25 μm. (Right) Quantitative data are shown. P value was determined by a two-sided Mann–Whitney U-test. Source data are provided as a Source data file.
