Figure-S1 from <i>ASS1</i> as a Novel Tumor Suppressor Gene in Myxofibrosarcomas: Aberrant Loss via Epigenetic DNA Methylation Confers Aggressive Phenotypes, Negative Prognostic Impact, and Therapeutic Relevance
Authors: Huang Hsuan-Ying , Wu Wen-Ren , Wang Yu-Hui , Wang Jun-Wen , Fang Fu-Min , Tsai Jen-Wei , Li Shau-Hsuan , Hung Hsiao-Chin , Yu Shih-Chen , Lan Jui , Shiue Yow-Ling , Hsing Chung-His , Chen Li-Tzong , Li Chien-Feng 
Published: April 2023

<p>Figure-S1 PDF file 194K,Figure-S1 Molecular basis of ASS1 deficiency in myxofibrosarcoma: (A) High-resolution oligonucleotide-based array comparative genomic hybridization shows normal DNA copy number at loci spanning ASS1 gene at 9q34.1 in the vast majority (14/15, 93.3%) of myxofibrosarcoma tissue and cell line samples, except for one featuring hemizygous deletion. In contrast, there are frequent DNA losses, including homozygous deletions near 9p21, on 9p in myxofibrosarcoma, which almost involve the whole chromosomal arm. (B) Quantitative pyrosequencing ascertains ASS1 methylation status detected by methylation-specific PCR. (a) Cell lines samples display results concordant with those detected by methylation-specific PCR. High degree of ASS1 promoter methylation is seen in NMFH-1 (left upper) and OH931 (right upper) myxofibrosarcoma cells, with the percentage of methylation >50% as shown at most representative CpG islands. Despite barely detectable ASS1 expression at the mRNA and protein levels in Figure-1B, there are comparatively less methylated CpG islands in NMFH-2 cells by pyrosequencing (data not shown), corresponding to presence of both methylated and unmethylated bands with similar intensity by methylation-specific PCR in Figure-1C. In contrast, there is no remarkable DNA methylation found in CCD966SK fibroblasts (left lower) and LPS510 dedifferentiated liposarcoma cells (right lower). (b) Formalin-fixed tissue samples of 10 cases with informative methylation-specific PCR data are selected for confirmatory pyrosequencing. Representative cases of highly methylated (MFS7, left) and hypomethylated (MFS25, right) tumors in pyrosequencing are illustrated, which show only methylated or unmethylated bands by methylation-specific PCR, respectively. Both methods of methylation assessment reveal concordant results in general, as summarized in Table-S2. (C) ASS1 mRNA expression in NMFH-1 cells is not restored with separate or combined use of trichostatin, 3-Dznep, UNC0638, and/or EZH2 knockdown. (a) By quantitative RT-PCR, neither 3-Dznep nor UNC0638 could recover ASS1 mRNA in NMFH-1 cells, regardless of the presence or absence of trichostatin and/or 5-aza-2��-deoxycytidine. 3-Dznep is a novel inhibitor that suppresses H3K27 histone methyltransferation mediated by polycomb recessive complex 2-associated EZH2, while UNC0638 is an inhibitor that targets G9a-mediated H3K9 histone methyltransferation. (b) Similar to 5-aza-2��-deoxycytidine alone, ASS1 mRNA expression level in NMFH-1 still remains barely detectable when the cells are alternatively or additionally treated with trichostatin, irrespective of the presence or absence of stable silencing of EZH2 expression by shEZH2. Inset: Western blotting and RT-PCR cross validate successful suppression of EZH2 expression with RNA interference. Histone H2B nuclear protein, shLacZ, and POLR2A transcript are run in parallel as the controls in Western blotting, RNA interference, and quantitative RT-PCR assays, respectively</p>