Fig 1: TCIRG1 knockdown has anti-tumorigenic effects on hepatocellular carcinoma (HCC) cells. (a) qRT-PCR analysis of TCIRG1 in 14 hepatic cell lines, including two normal (THLE3 and MIHA) and 12 hepatoma cell lines (Hep3B, HepG2, Huh7, PLC/PRF/5, SK-Hep1, SNU354, SNU368, SNU387, SNU398, SNU423, SNU449 and SNU475). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization (mean±s.d.; n=3, **P<0.01; ***P<0.001). (b) Endogenous protein expression of TCIRG1 in hepatic cell lines was analyzed by western blotting. GAPDH was used as a loading control, and the numbers under the blot indicate the relative expression level of each protein. (c) Clonogenic assays were performed in SNU475 and Huh7 cell lines. Upper panel: representative images of colonies. Lower panel: the data from three randomly selected images shown in the form of a graph (mean±s.d.; n=3, *P<0.05, **P<0.01). (d) SNU475 and Huh7 cell lines were transfected with negative-control siRNA or TCIRG1-specific siRNA. Cell number was determined using trypan blue-based cell counting (mean±s.d., n=3, **P<0.01). (e) SNU475 and Huh7 cell lines were transfected with negative-control siRNA or TCIRG1-specific siRNA. Cell growth was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. (f) Bromodeoxyuridine (BrdU) incorporation assays were carried out using SNU475 and Huh7 cell lines (mean±s.d.; n=3, *P<0.05, **P<0.01). (g) Fluorescence-activated cell sorting (FACS) analysis was conducted after negative-control siRNA or TCIRG1-specific siRNA transfection. When TCIRG1 was knocked down, G1/S arrest was observed in SNU475 and Huh7 cell lines. The percentage indicates the distribution of cells in the different phases of cell cycle (mean±s.d., n=3, *P<0.05). (h) Negative-control siRNA (NC) or TCIRG1-specific siRNA (siTCIRG1) were transfected into cells. FACS analysis was performed using PI and Annexin V staining. The bar graphs indicate the percentage of annexin V-positive cells (mean±s.d., n=3, *P<0.05). (i) Following transfection, cells were treated with 3-MA (5 mM), and FACS analysis was performed. The bar graph indicates the percentage of Annexin V negative and PI-positive cells (mean±s.d., n=3, ***P<0.001).
Fig 2: Effect on Tcirg1 knockdown in an experimental model of metastasis. (a) Representative CT image of ras-transformed NIH-3T3 cells after transient suppression of Tcirg1 (left). The volume of each tumor was calculated, and the total tumor volume was determined (right). The arrows indicate tumor mass. (b) In vivo mages of ras-transformed NIH-3T3 cells using a lung metastasis mouse model after transfection with negative-control siRNA (n=5) and Tcirg1-targeting siRNA (n=5) (left). The number of nodules in mouse lungs is shown by the bar graph (right) (mean±s.d., n=5, ***P<0.001).
Fig 3: TCIRG1 knockdown attenuates the metastatic potential of hepatocellular carcinoma (HCC) cells. (a, b) TCIRG1 knockdown inhibited migration (a) and invasion (b) of SNU475 and Huh7 cell lines in vitro. The number of migrated and invaded cells was determined. Three randomly selected fields were captured, and the results were graphically presented (mean±s.d., n=3, **P<0.01, ***P<0.001). Representative images are shown. (c) Wound healing assay. The bar graphs show the ratios of the recovered area (mean±s.d., n=3, ***P<0.001). (d, e) Effect of TCIRG1 knockdown on cell migration and invasion in vitro. TCIRG1 knockdown inhibited migration (d) and invasion (e) of ras-transformed NIH-3T3 mouse fibroblasts. The number of migrated and invaded cells was determined. Three randomly selected fields were captured, and the results were presented in the form of a graph (mean±s.d., n=3, **P<0.01). Representative images. (f) Western blot analysis of epithelial–mesenchymal transition (EMT) markers. The protein level of TCIRG1, E-cadherin, N-cadherin, Fibronectin, Vimentin, Snail and Slug was detected with their specific antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. The numbers under the blot indicate the relative expression level of each protein. (g) Analysis of the NCBI GEO database. GEO data sets with the accession numbers GSE39791 and GSE77314 were used for expression analysis of TCIRG1 and CDH1. Left panel, CDH1 expression in GSE39791 and GSE77314. Right panel, negative correlation of TCIRG1 and CDH1 expression in GSE39791 (Pearson’s correlation coefficient, r=-0.21, **P<0.01) and in GSE77314 (Pearson’s correlation coefficient, r=-0.36, ***P<0.001).
Fig 4: Validation of TCIRG1 overexpression in hepatocellular carcinoma (HCC) and its clinical relevance. (a) Analysis of TCGA_LIHC data. TCGA_LIHC data showed that TCIRG1 was significantly overexpressed in HCC tissues compared with that in non-tumor tissues in all the analyzed samples (n=423, left) and paired samples (n=100, middle), and TCIRG1 was overexpressed in higher tumor grade (right) (mean±s.d.; *P<0.05; ***P<0.001). (b) Kaplan–Meier survival analysis of TCIRG1 mRNA expression in TCGA_LIHC for overall survival (left) and disease-free survival (right). The P-value was obtained with the log-rank test. (c) Receiver operating characteristic curve (ROC) analysis of TCIRG1 in TCGA_LIHC (left) and GSE39791 (right). Statistically significant difference of the area under the curve (AUC) compared with reference line (*P<0.05; ***P<0.001). (d) The mRNA expression of TCIRG1 was determined by qRT-PCR in a subset of HCC tissues. (e) Representative photomicrographs of immunohistochemical analysis of non-tumor or tumor tissues (left) and extent of TCIRG1 expression (right) (***P<0.001). (f) Western blot analysis of TCIRG1 in a subset of HCC tissues (NT: adjacent non-tumor tissues; T: tumor tissues). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. (g) Kaplan–Meier survival curves (left) for patients with positive or negative expression of TCIRG1, and the extent of TCIRG1 expression (right) in the tumors of the non-transplanted cohort. (h) Kaplan–Meier survival curves (left) for patients with positive or negative expression of TCIRG1, and the extent of TCIRG1 expression (right) in the tumors of the transplantation cohort.
Supplier Page from Abcam for Anti-TCIRG1 antibody