Fig 1: The key enzymes of the urea cycle were often low expressed in HCC patients. (A) UALCAN portal analysis of cancer samples from the TCGA database (http://ualcan.path.uab.edu/). A comparison of CPS1 expression between normal and multiple cancer samples. Tumor tissues were shown in red, and normal tissues were shown in blue. BLCA, Bladder urothelial carcinoma; BRCA, Breast invasive carcinoma; CESC, Cervical squamous cell carcinoma; CHOL, Cholangiocarcinoma; COAD, Colon adenocarcinoma; ESCA, Esophageal carcinoma; GBM, Glioblastoma multiforme; HNSC, Head and Neck squamous cell carcinoma; KICH, Kidney Chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; PAAD, Pancreatic adenocarcinoma; PRAD, Prostate adenocarcinoma; PCPG, Pheochromocytoma and Paraganglioma; READ, Rectum adenocarcinoma; SARC, Sarcoma; SKCM, Skin Cutaneous Melanoma; THYM, Thymoma; STAD, Stomach adenocarcinoma; UCEC, Uterine Corpus Endometrial Carcinoma. (B) Expression of CPS1 of normal liver (N) and HCC (T) samples from The Human Protein Atlas (Human Protein Atlas available from http://www.proteinatlas.org). (C) UALCAN portal analysis of CPS1 expression between normal and different grade HCC samples from the TCGA database. (D) Western blot to detect the expression of CPS1, OTC, ARG1 and GAPDH from 14 pairs of cancer and adjacent tissues of HCC. (E,F) Survival probability between HCC patients with high and low CPS1 expression. The GEPIA database (http://gepia.cancer-pku.cn/) and Kaplan-Meier Plotter (http://kmplot.com/analysis/) were used to conduct survival analyses based on core gene expression. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 2: The relationship of urea cycle with ammonia metabolism in cancer cells. (A) Western blot of lysates from HL-7702 cells cultured under 0, 0.5, 2, 5, 10 or 20 mM NH4Cl for 8 h. (B) Western blot of lysates from HepG2, NCM460, HCT116, MHCC97H, HLE, MCF-7, PLC/PRF/5 cells cultured under 0 or 10 mM NH4Cl for 8 h. (C,D) Relative urea excretion from HL-7702 and PLC/PRF/5 cells cultured under 0, 0.5, 2, 5, 10 or 20 mM NH4Cl for 48 h. (E) Western blot confirmed the knockdown of CPS1 in PLC/PRF/5 cells. (F) Relative ammonia excretion in PLC/PRF/5/shScramble, PLC/PRF/5/shCPS1 for 8 hours. (G) Relative urea excretion in PLC/shScr, PLC/shCPS1 for 48 h. (H) Western blot confirmed the over-expression of CPS1 in MHCC97H cells. (I) Relative ammonia excretion in MHCC97H/Vector, MHCC97H/CPS1 for 8 h. (J) Relative urea excretion in MHCC97H/Vector, MHCC97H/CPS1 for 48 h. Values are the means ± SD of three independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 3: Urea cycle protects cancer cells from high concentrations of ammonia. (A) Colony formation ability of HL-7702, PLC/PRF/5, HepG2 and MHCC97H under different concentrations of NH4Cl. The culture time was 2 weeks. (B–E) Colony number quantification of HL-7702, PLC/PRF/5, HepG2 and MHCC97H under different concentrations of NH4Cl by Image J. (F) Proliferation of PLC/PRF/5/shScr and PLC/PRF/5/shCPS1 cells cultured under control or 10 mM NH4Cl for 3 days. (G) Proliferation of HepG2/shScr and HepG2/shCPS1 cells cultured under control or 10 mM NH4Cl for 3 days. (H) Proliferation of MHCC97H/Vector, MHCC97H/CPS1 cells cultured under control or 10 mM NH4Cl for 3 days. (I) Proliferation of HCT116/Vector, HCT116/CPS1 cells cultured under control or 10 mM NH4Cl for 3 days. Values are the means ± SD of three independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.
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