Journal: Experimental & Molecular Medicine

Article Title: T-cell immune regulator 1 enhances metastasis in hepatocellular carcinoma

doi: 10.1038/emm.2017.166

Figure Lengend Snippet: 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 m M ), 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).

Article Snippet: Hep3B, HepG2, Huh7, PLC/PRF/5, SK-Hep1, SNU354, SNU368, SNU387, SNU398, SNU423, SNU449 and SNU475 HCC cell lines were acquired from the Korean Cell Line Bank (KCLB, Seoul, South Korea).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Control, Transfection, Negative Control, Cell Counting, BrdU Incorporation Assay, Fluorescence, FACS, Staining

Journal: Experimental & Molecular Medicine

Article Title: T-cell immune regulator 1 enhances metastasis in hepatocellular carcinoma

doi: 10.1038/emm.2017.166

Figure Lengend Snippet: 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).

Article Snippet: Hep3B, HepG2, Huh7, PLC/PRF/5, SK-Hep1, SNU354, SNU368, SNU387, SNU398, SNU423, SNU449 and SNU475 HCC cell lines were acquired from the Korean Cell Line Bank (KCLB, Seoul, South Korea).

Techniques: Knockdown, Migration, In Vitro, Wound Healing Assay, Transformation Assay, Western Blot, Control, Expressing

Journal: Aging Cell

Article Title: Liver osteopontin is required to prevent the progression of age‐related nonalcoholic fatty liver disease

doi: 10.1111/acel.13183

Figure Lengend Snippet: Liver OPN is p53 regulated. (a) Protein levels of p53 were evaluated by immunohistochemistry in liver sections of 3‐, 10‐, and 20‐month‐old (m) wild‐type (WT) mice ( n = 3–5). (b) OPN protein levels from liver homogenates were measured in WT and p53‐KO male and female mice fed a chow diet (CD) and a high‐fat diet (HFD) by immunoblotting using glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as loading control ( n = 4–6). (c) Liver p53 and OPN protein levels were evaluated in WT mice fed a HFD injected with p53‐dominant positive adenovirus (adp53) and GFP (adGFP), using transferrin or GAPDH as a loading control ( n = 4–6). (d) Protein levels of p53 and OPN in siCtrl and sip53 HepG2 cells were measured by immunoblotting using GAPDH as loading control. Extracellular OPN was measured using an ELISA ( n = 4–5). (e) OPN protein levels from HepG2 cells silenced for p53 and Hep3B cells treated with palbociclib (Palbo) were measured by immunoblotting using GAPDH as loading control. OPN media levels from Hep3B cells treated either with vehicle (Veh) or with palbociclib (Palbo) were measured by ELISA ( n = 4–8). Values are means ± SEM . Significant differences are denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001 (Student's t test)

Article Snippet: Data obtained from the Cell Line Encyclopedia (© 2019 The Broad Institute of MIT & Harvard) showed that the cell line with the least p53 expression, the Hep3B, also had the lowest OPN expression.

Techniques: Immunohistochemistry, Western Blot, Control, Injection, Enzyme-linked Immunosorbent Assay