Fig 1: Overexpression of ARG1 augments EMT and the migration ability of cancer cells. GFP-transfected CT26 mock control and CT26 Arg1 OE cells were established using the pMX-IRES-GFP vector. A Gene expression levels of Arg1 were investigated by qPCR. The mean values and SDs (n = 4) are indicated. *P < 0.05 by Student’s t-test. B ARG1 protein expression levels were evaluated by Western blotting. The mean values and SDs (n = 3) are indicated. *P < 0.05 by Student’s t-test. C Intracellular ARG1 activity was determined by EIA. The mean values and SDs (n = 4) are shown. *P < 0.05 by Student’s t-test. D GFP-transfected CT26 mock control and CT26 Arg1 OE cells (5 × 106) were cultured for 24 h. Intracellular levels of l-Arginine, l-ornithine, l-citrulline, and urea were evaluated by HPLC, and the mean values and SDs (n = 4, three independent experiments) are shown. *P < 0.05 by Student’s t-test. E GFP-transfected CT26 mock control and CT26 Arg1 OE cells (5 × 104) were cultured for 24 h. Migration ability was evaluated by Transwell assay at 24 h. Representative images are indicated. Bars represent 200 μM. The mean values and SDs (n = 4) are indicated. *P < 0.05 by Student’s t-test. F N-cadherin and E-cadherin protein expression levels were determined by Western blotting. Representative images are indicated. The mean values and SDs (n = 3) of the relative expression levels against α-tubulin are indicated. *P < 0.05 by Student’s t-test
Fig 2: ARG1 activity and gene expression are related to malignancy in human colon cancer. A ARG1 protein expression levels in normal (N = 28) and tumor (N = 42) tissues of CRC patients are shown according to the CPTAC database. *P < 0.05 by unpaired t-test. B Arg1 gene expression levels in primary tumor (N = 18) and liver metastasis (N = 18) of CRC patients were analyzed according to the data from the GEO dataset (GSE14297). *P < 0.05 by unpaired t-test. C HCT116 cells (5 × 103) were cultured in the absence and presence of nor-NOHA (0, 125, 250, 500 μM). Living cell numbers were evaluated at 12 h and 24 h. The mean and SD values (n = 4) are indicated. *P < 0.05 by Student’s t-test. D HCT116 cells (2 × 104) were cultured in the absence and presence of nor-NOHA (0, 125, 250, 500 μM). Migration ability was evaluated by Transwell assay at 24 h. Representative images are indicated. Bars represent 200 μM. The mean and SD values (n = 4) are indicated. *P < 0.05 by Student’s t-test
Fig 3: Overexpression of ARG1 augments the metastatic colonization ability of cancer cells. A GFP-transfected CT26 mock control and CT26 Arg1 OE cells (2 × 105) were intravenously or intrasplenically inoculated into mice. Sera and liver or lung tissues for assay were collected on day 14. B Serum l-arginine levels were evaluated by HPLC, and the mean values and SDs (n = 4, three independent experiments) are shown. C Serum arginase activity was determined by EIA, and the mean values and SDs (n = 4–5) are shown. D Metastatic colonization in the liver tissue of mice was evaluated using an in vivo imaging system on day 14. Representative images of normal liver and GFP-expressing CT26 cell-bearing livers are shown. Photon flux ratios were determined from the images of liver metastatic colonization model mice (n = 4, three independent experiments). E HE staining of liver tissue was performed 14 days after inoculation. Representative images are shown. Bars in the images represent 200 mm. F Metastatic colonization in the lung tissue of mice was evaluated using an in vivo imaging system at day 14. Representative images of normal liver and GFP-expressing CT26 cell-bearing livers are shown. Photon flux ratios were determined from images of the liver metastatic colonization model mice (n = 4, three independent experiments). G HE staining of lung tissue was performed 14 days after inoculation. Representative images are shown. Bars in the images represent 500 mm. Ratios of the tumor area relative to the total liver tissue area were calculated by the ImageJ software (E, G). The mean and SD values from four independent mice are shown
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