Crystal-to-Crystal Solid-State Photochemistry: Absolute Asymmetric β-Thiolactam Synthesis from an Achiral α,β-Unsaturated Thioamide

Crystal-to-Crystal Solid-State Photochemistry:  Absolute Asymmetric β-Thiolactam Synthesis from an Achiral α,β-Unsaturated Thioamid

Bisulfite sequencing of the <i>Apc</i> promoter region in small intestinal tumors of B6/B6-Chr18^MSM-F1 <i>Apc</i>^<i>Min/+</i> mice.

Methylation status of the 93 CpG dinucleotides in the promoter region of Apc. Each circle represents a CpG site in the genomic DNA sequence and each row of circles represents the analysis of a single cloned allele. Closed circles: methylated CpG dinucleotides; open circles: unmethylated CpG dinucleotides. Bisulfite efficiency averaged 98.5 ± 1.33% for CpG island 1 and 98.8 ± 1.23% for CpG island 2 in all samples examined.</p

Sequence analysis of <i>Apc</i> in intestinal tumors from B6/B6-Chr18^MSM-F1 <i>Apc</i>^<i>Min/+</i> mice.

Sequence analysis of Apc in intestinal tumors from B6/B6-Chr18MSM-F1 ApcMin/+ mice.</p

Analysis of LOH of Chr18 in mouse intestinal tumors.

Each row shows the LOH of one microsatellite marker on Chr18, and each column shows the data for one intestinal tumor. Open circles indicate the loss of either the wild-type or Min alleles, and filled circles indicate the retention of both alleles. Based on the LOH results, each tumor was categorized as 0 Gy Unidentified, 2 Gy Unidentified, or 2 Gy Deletion. (TIF)</p

Mutations identified in the Unidentified type of intestinal tumors.

Mutations identified in the Unidentified type of intestinal tumors.</p

Supplemental Figure Legends from Radiation-Induced Myofibroblasts Promote Tumor Growth via Mitochondrial ROS–Activated TGFβ Signaling

S1. The percentage of TIG-3 and MRC-5 cells with alpha-SMA staining exposed to indicated doses is shown in the graph. S2. Tumor volumes of HeLa, HeLa+MRC-5 0FR tumors, HeLa+MRC-5 0.05FR tumors, HeLa+MRC-5 NAC 0FR tumors, and HeLa+MRC-5 0.05FR tumors.</p

Bisulfite sequencing of the sites corresponding to human <i>APC</i> promoter 1A in small intestinal tumors of B6/B6-Chr18^MSM-F1 <i>Apc</i>^<i>Min/+</i> mice.

Methylation status of the 21 CpG dinucleotides in the promoter region of Apc. Each circle represents a CpG site in the genomic DNA sequence and each row of circles represents the analysis of a single cloned allele. Closed circles: methylated CpG dinucleotides; open circles: unmethylated CpG dinucleotides. (TIF)</p

<i>Apc</i> genomic region analyzed in this study.

Nucleotide positions are numbered relative to the transcription start site of Apc (NM_007462). Capital letters represent CpG islands obtained from the UCSC Genome Browser (http://genome.ucsc.edu/). The CpG sites are highlighted in red. Primer pairs (boxed sequences) were designed using MethPrimer (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi). Arrows indicate the direction from 5’ to 3’. Due to the relative length of this CpG island, it was split into two segments, and bisulfite sequencing was performed for each segment. (TIF)</p

Primers and PCR conditions.

Among the small intestinal tumors that occur in irradiated mice of the established mouse model B6/B6-Chr18MSM-F1 ApcMin/+, loss of heterozygosity analysis can be utilized to estimate whether a deletion in the wild-type allele containing the Adenomatous polyposis coli (Apc) region (hereafter referred to as Deletion), a duplication in the mutant allele with a nonsense mutation at codon 850 of Apc (Duplication), or no aberration (Unidentified) has occurred. Previous research has revealed that the number of Unidentified tumors tends to increase with the radiation dose. In the present study, we investigated the molecular mechanisms underlying the development of an Unidentified tumor type in response to radiation exposure. The mRNA expression levels of Apc were significantly lower in Unidentified tumors than in normal tissues. We focused on epigenetic suppression as the mechanism underlying this decreased expression; however, hypermethylation of the Apc promoter region was not observed. To investigate whether deletions occur that cannot be captured by loss of heterozygosity analysis, we analyzed chromosome 18 using a customized array comparative genomic hybridization approach designed to detect copy-number changes in chromosome 18. However, the copy number of the Apc region was not altered in Unidentified tumors. Finally, gene mutation analysis of the Apc region using next-generation sequencing suggested the existence of a small deletion (approximately 3.5 kbp) in an Unidentified tumor from a mouse in the irradiated group. Furthermore, nonsense and frameshift mutations in Apc were found in approximately 30% of the Unidentified tumors analyzed. These results suggest that radiation-induced Unidentified tumors arise mainly due to decreased Apc expression of an unknown regulatory mechanism that does not depend on promoter hypermethylation, and that some tumors may result from nonsense mutations which are as-yet undefined point mutations.</div

Analysis of <i>Apc</i> mRNA expression in the small intestinal tumors of B6/B6-Chr18^MSM-F1 Apc^<i>Min/+</i> mice.

Quantitative PCR was performed using Gapdh as an endogenous control. Each point represents Apc mRNA level in an individual tumor (0 Gy Unidentified, n = 14; 2 Gy Unidentified, n = 15; 2 Gy Deletion, n = 6; normal intestinal tissue, n = 4). Primers were set to span exons 7 and 8 (A) and exons 16 and 17 (B) according to the NCBI database (NM_001402727.1). The means and standard deviations are represented by bars and error bars, respectively. Apc expression in normal intestinal tissue was set to 1. The triangle among the 2 Gy-Unidentified data points represents Apc expression in Tumor ID 2-2-23, in which a deletion comprised approximately 3.5 kbp at the end of the last exon of Apc. ** p (TIF)</p