Fig 2: Emodin can directly bind to NCOR2. (A) Effect of concentration of emodin and emodin-biotin (B-Emodin) on transcription of SerRS expressed as firefly/renilla ratio (n=3). (B) The protein level of SerRS with emodin (10 µM) and B-Emodin (10 µM). (C) SDS-PAGE of MDA-MB-231 cell lysates incubated with free biotin or B-Emodin following protein affinity pull-down and coomassie blue staining (n=3). Arrows indicate bands analyzed by mass spectrometry (MS). (D) Immunoblot with anti-NCOR2, anti-ACACA, anti-FASN, anti-CAD, and anti-a-Tubulin antibodies of protein precipitated by streptavidin beads from MDA-MB-231 cell lysates in the presence of B-Emodin or biotin (10 µM). (E) shRNA target human NCOR2 was transfected into MDA-MB-231 cells, NCOR2 expression in MDA-MB-231 cells was determined by qPCR after shRNA transfection. (F) mRNA levels of SerRS from MDA-MB-231-shLacZ and MDA-MB-231-shNCOR2 cells treated with emodin for 48 h (n=3). (G) Representations of the predicted binding modes of emodin with NCOR2, SANT2. (H) Immunoblot with anti-NCOR2 antibody of purified proteins (GST and GST-NCOR2 SANT2) incubated with B-Emodin (10 µM) or biotin and precipitated by streptavidin beads. Data are means ± SD., ns, no significant difference; **p<0.01; ***p<0.001; by one-way ANOVA for A; by Student's t-test for E, F.

Fig 3: Nucleotide de novo synthesis genes are up-regulated in metastatic breast cancer cells and reversely correlate with patient’s survival.A. Heatmap of differentially expressed genes from RNA-seq data showed that 25 genes that regulated nucleotide de novo synthesis were up-regulated in the 4TO7Lung cells and 8 genes that regulated salvage synthesis were down-regulated. B. GSEA of KEGG pathway showed that the genes up-regulated in the 4TO7Lung cells are enriched in the purine and pyrimidine metabolism pathways. NESs and q values are shown. The raw data can be found in S1 Raw images. C. The protein expression levels of selected genes of de novo synthesis in 4T1, 4TO7Ori, and 4TO7Lung cells. D. Relative mRNA levels of nucleotide de novo synthesis related genes in 4T1, 4TO7Ori, and 4TO7Lung cells (n = 3 replications). E. Relative mRNA levels of Prps2 in a variety of human breast cancer cell lines (n = 3 replications). F. PRPS2 expression in a variety of human cell lines. G. Relative mRNA levels of Prps2 in a variety of murine breast cancer cell lines (n = 3 replications). H. PRPS2 expression in a variety of murine cell lines. I. HE staining and IHC analyses of nucleotide de novo synthesis proteins in primary and metastatic foci of MMTV-PyMT mice. Scale bars, 160 µm for low magnification and 40 µm for high magnification. J. Kaplan–Meier survival analysis of breast cancer patients (n = 542) with different levels of genes in purine de novo synthesis pathway. Information of patients was acquired from TCGA database. K. Kaplan–Meier survival analysis of breast cancer patients (n = 542) with different levels of genes in pyrimidine de novo synthesis pathway. Information of patients was acquired from TCGA database. All values are presented as means ± SD. Statistical significance is shown as *P = 0.05, **P = 0.01, and ***P = 0.001. The raw data used for quantification of D, E, G, J, and K can be found in S2 Data. ADSS, adenylosuccinate synthetase; CAD, carbamoyl-phosphate synthetase 2; FDR, false discovery rate; GART, phosphoribosylglycinamide synthase; GSEA, gene set enrichment analysis; GMPS, guanine monophosphate synthase; IHC, immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes; MMTV-PyMT, mouse mammary tumor virus-polyoma middle tumor-antigen; NES, normalized enrichment score; PFAS, phosphoribosyl formylglycinamidine synthase; PRPS2, phosphoribosylpyrophosphate synthetase 2; TCGA, The Cancer Genome Atlas.

Fig 4: (A)Typical images of IHC in 16 pairs of colon cancer tissues showing the protein expression of ASNS, CEBPA and CAD in colon cancer tissues and adjacent nontumor tissues. Up panel, scale bar 1000 µm; down panel, scale bar 100 µm. (B) Immunohistochemical positive cell rates of ASNs, CEBPA, and CAD in 7 colon cancer tissues and adjacent nontumor tissues in group 1. (C) Immunohistochemical positive cell rates of ASNS, CEBPA and CAD in 9 colon cancer tissues and adjacent nontumor tissues in group 2. *p < 0.05, **p < 0.01 and ***p < 0.001, analyzed using unpaired t-test. Group 1, n = 7; Group 2, n = 9.

Fig 5: (A) ASNS, CEBPA, and CAD expression levels in Case 1 and Case 16 of tumor specimens and adjacent normal specimens. (B) The relative gray values of western blot, which represent the relative protein expression level, of ASNS, CEBPA, and CAD in 7 Colon cancer tissues and adjacent nontumor tissues in (B) Group 1 and (C) Group 2.*p < 0.05, **p < 0.01 and ***p < 0.001, analyzed using unpaired t-test. Group 1, n = 7; Group 2, n = 9.