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. 2022 Mar 4;257(1):39–52. doi: 10.1002/path.5868

Figure 5.

Figure 5

Characterization of RNF43 mutations in CRC cells. (A) Schematic drawing of the experimental strategy. C14, C24, and C45 RNF43 frameshift mutant organoids were subjected to subcloning (n = 6 subclones for each line); RNF43 mRNAs were amplified by RT‐PCR, and cDNAs were subcloned to the plasmid (n = 7–12 cDNAs for each subclone). RNF43 mutation patterns were then examined by sequencing. (B) The RT‐PCR strategy to examine cis or trans mutations of p.Pro370fs and p.Gly659fs by the primer F1 and R1, and p.Arg225fs and p.Pro370fs by the primer F2 and R2. Arrows indicate the locations of the respective primers. Closed bars indicate expected amplified cDNA fragments by the primers. (C) Representative sequencing results of wild‐type (top) and mutant RNF43 (bottom) for p.Arg225fs (left), p.Pro370fs (center), and p.Gly659fs (right) positions. Note that the mutations cause amino acid changes (blue). (D) Schematic drawing of RNF43 allele types identified by sequencing in the C14 and C24 subclones (left, top) and C45 subclones (left, bottom). The frequencies of the respective allele types in each subclone are indicated as color bars for C14 (right, top), C24 (right, middle), and C45 (right, bottom). Asterisks indicate subclones used for in vitro and xenograft drug dosing experiments (Figure 6 and supplementary material, Figures S3 and S4). The numbers indicated below the bar graphs indicate the total number of independently amplified and sequenced cDNAs. (E) List of possible RNF43 genotypes of the parental organoid lines. (F) The results of TaqMan SNP genotyping at RNF43 codon 370 (wild‐type: HEX versus p.Pro370fs: FAM) for C14 and C24 subclones (purple diamonds) and conventional pathway‐type CRC organoids (green squares) are shown as a scatter plot with RNF43 pPro370fs cDNA control (blue circle) and no template control (NTC) (light‐green triangle). (G) Top: the frequencies of transposon insertions in the Rnf43, Znrf3, and Apc genes in SB‐induced intestinal tumors. Bottom: the locations of transposon insertions in the Rnf43 gene. Exons including R225, P370, and G659 are indicated by arrows.