Fig 2: Expression of EMT-related transcripts and proteins in ovine conceptuses during the peri-implantation period. (A) RT-qPCR analysis of transcripts encoding EMT-related factors in P15, P17, and P21 ovine conceptuses. RNA was extracted from frozen conceptuses (n = 3/day). Data relative to the value of P15 are shown. Values represent the means ± SEM. ACTB was used as the reference gene. *** P < 0.001, * P < 0.05. (B) Western blot analysis of EMT-related proteins in P15, P17, and P21 ovine conceptuses (n = 3/day). While VIM and CDH2 are expressed only on P21, some epithelial markers, such as cytokeratin and CDH1, are expressed on P21. The numbers on the left denote the molecular weight markers, while those on the right are the product sizes. ACTB was used as the loading control.

Fig 3: Generation of triple-knockout mice and their phenotypes. Approximately 4 pl of RNA solution containing 10 ng/μl each of Cas9 mRNA, Tyr gRNA, Gdf8 gRNA and Hprt gRNA was injected into the cytoplasm of each one-cell embryo and cultured in M16 medium. Sixty 2-cell embryos were transferred into the oviductal ampullae (10 embryos per oviduct) of 3 recipients. (A) Generation efficiency of triple-knockout mice. (B) Three examples of the 8 pups (#1-#3 in Supplementary Fig. 1) with a white coat color over the whole body. (C) One of the 9 pups (#8 in Supplementary Supplementary Fig. 1) showed a black and white striped mosaic coat pattern. (D) Immunoblotting of Hprt and beta-Actin using a muscle sample from an Hprt biallelic knockout female pup (#1 in Supplementary Fig. 1). (E) Hyperproduction of skeletal muscle in a knockout mouse (KO, #1 in Supplementary Fig. 1) compared with a wild-type mouse (WT).

Fig 4: (A) Western blot of H3S10P, H3K27me3, and β-actin. The following commercially available antibodies were used at the indicated concentrations: H3S10P (05-806, Millipore, 1:50,000), H3K27me3 (GTX12184, Genetex, 1:1,000), and β-actin (GTX109639, Genetex, 1:12,500). Lysates for western blotting were made from five worms that were collected at each time point. (B) SA-β-gal activity staining in irradiated worm tissues. Black arrows point to positive staining for SA-β-gal activity.

Fig 5: SOD2high/MCTS1high expression is a new poor prognostic marker in invasive breast carcinoma.A, B The relapse-free survival (RFS) of breast cancer patients classified by low (n = 2947) and high (n = 1004) MCTS1 expression (A) or by low (n = 1449) and high (n = 2502) SOD2 expression (B) was evaluated using Kaplan–Meier (KM) Plotter analysis. C The correlation of MCTS1 and SOD2 gene expression in breast cancer patients (n = 159) was analyzed using the Pawitan dataset in the Oncomine cancer profiling database. D–F SOD2 expression was analyzed in TissueScan cDNA arrays with normal breast tissue (n = 11) and breast tumor biopsies (n = 124) across distinctive tumor stages (D, E), molecular subtypes and TNM classifications (F). The SOD2 mRNA levels in tumor samples were normalized to internal ACTB (ß-actin) mRNA levels and then compared with the levels in normal breast samples. Data are presented as the mean ± s.e.m. G KM Plotter was used to estimate the survival of patients with MCTS1high/SOD2high (n = 37) expression compared with that of those with MCTS1low/SOD2low (n = 60) expression using the Pawitan dataset in the Oncomine database. H, I RFS was estimated for ER-negative (n = 434) (H) and basal-like (n = 309) (I) breast cancer patients with MCTS1high/SOD2high or MCTS1low/SOD2low expression using KM Plotter. Statistical analysis was performed using the log-rank Mantel–Cox test (A, B, and G–I), Pearson product-moment correlation coefficient (C), Kruskal–Wallis test followed by Dunn’s multiple comparison test (D) and ?2 test (E, F).