thle3 (normal liver) cells  (POSTECH Inc)

Journal: Cell reports

Article Title: De novo sphingolipid biosynthesis necessitates detoxification in cancer cells

doi: 10.1016/j.celrep.2022.111415

Figure Lengend Snippet: (A) Heatmap of unsupervised hierarchical clustering analysis of the mRNA expression patterns of 2 KDSR KO-sensitive (DLD1 and NCIH838) and 1 -insensitive (HUH7) cancer cell line following CTRL or KDSR KO. Numbers at the end of each label are biological repeat identifier. Sensitive lines with KDSR KO are labeled in red. (B and C) Gene clusters that are upregulated (B, yellow) or downregulated (C, blue) specifically in DLD1 and NCIH838 KDSR KO (sensitive) cells. Tables list examples of Gene Ontology (GO) biological processes that are significantly enriched in each gene cluster. (D) Western blot showing protein levels of characteristic markers of ER stress in DLD1 cells subjected to CTRL or KDSR g1- or g2-mediated KO at 9 days after lentiviral transduction. DLD1 cells treated with 2 μM tunicamycin for 24 h were used as a positive control for ER stress induction. (E) Top row, representative electron microscope images of DLD1 CTRL and KDSR KO cells, depicting an overview of the aberrant morphology observed in KDSR KO cells. N indicates nucleus; scale bar: 3 μm. Bottom row, higher magnification images displaying specific features of the subcellular structures in KDSR KO cells. Red arrows point to areas where ribosomes line the edges of the structures. Left image scale bar: 0.2 μm, right image scale bar: 0.1 μm. (F) Immunofluorescent images of CTRL and KDSR KO cells stained for calnexin (ER membrane, red), phalloidin (actin, green), and Hoechst 33342 (nucleus, blue). Bottom rows show zoomed-in view of ER structures. Top row scale bar: 10 μm, bottom row scale bar: 4 μm. (G) Western blot of K48 ubiquitinated proteins in DLD1 cells treated with 1 or 10 μM of the proteasome inhibitor MG132 (18 h) or with KDSR KO with g1 or g2 (10 days) compared with vehicle and CTRL conditions, respectively. (H) Representative immunofluorescent images of DLD1 cells subjected to CTRL or KDSR KO or treated with vehicle or 3 μM MG132 and stained with aggresome dye (red), which binds beta-sheet structures found in protein aggregates. Nuclei were stained with DAPI (blue). Scale bar: 40 μm. (I) Fold change in protein levels of ER stress markers (IRE1a and BiP) and K48 ubiquitinated proteins in KDSR KO cells (average of g1 and g2) relative to CTRL cells in 3 non-cancer (GM05565, THLE3, CCD841CoN) and 3 sensitive cancer (DLD1, NCIH838, U251) cell lines. Each point represents an independent cell line as n = 3 replicates. Band intensities were quantified from westerns that are shown in (D) and (G) and and and were normalized to actin band intensity. For RNA sequencing, RNA was extracted from n = 3 biological replicates for each condition. GO enrichment analysis was performed using the PANTHER overrepresentation test. Data are shown as mean + SD. p values were calculated using two-tailed Student’s t test (*p < 0.05, **p < 0.01, ns = not significant).

Article Snippet: THLE3 (normal liver) cells were a kind gift of the Kwan Yong Choi Lab (POSTECH, Korea).

Techniques: Expressing, Labeling, Western Blot, Transduction, Positive Control, Microscopy, Staining, Membrane, RNA Sequencing, Two Tailed Test

Journal: International Journal of General Medicine

Article Title: microRNA-19a-3p and microRNA-376c-3p Promote Hepatocellular Carcinoma Progression Through SOX6-Mediated Wnt/β-Catenin Signaling Pathway

doi: 10.2147/IJGM.S278538

Figure Lengend Snippet: miR-19a-3p and miR-376c-3p are highly expressed in HCC tissues and cells. ( A ) Microarray-based analysis of differentially expressed miRNAs between HCC tissues and adjacent normal tissues. ( B ) miR-19a-3p and miR-376c-3p expression in 21 cases of HCC tissues and adjacent normal tissues tested by RT-qPCR. ( C ) miR-19a-3p and miR-376c-3p expression in human normal liver cell line THLE3 and HCC cell lines (Hep3B, MHCC-97L, Huh7) determined by RT-qPCR. ( D ) detection of miR-19a-3p expression in the TCGA database. ( E ) detection of miR-19a-3p expression at different stages of HCC. ( F ) analysis of the prognosis of patients with differential expression of miR-19a-3p. ( G ) detection of miR-376c-3p expression in the TCGA database. ( H ) detection of miR-376c-3p expression at different stages of HCC. ( I ) analysis of the prognosis of patients with differential expression of miR-376c-3p. The measurement data were depicted as mean ± standard deviation. The data between two groups were compared by unpaired t -test or and the data among multiple groups were compared by two-way ANOVA. The experiment was repeated three times. * p < 0.05 vs adjacent normal tissues or THLE3 cells.

Article Snippet: Human normal liver cell line THLE3 and HCC cell lines (Hep3B, American Type Culture Collection, Rockville, Maryland, USA; MHCC-97L and Huh7, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., Shanghai, China) were, respectively, seeded in Roswell Park Memorial Institute (RPMI)-1640 cell culture medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin.

Techniques: Microarray, Expressing, Quantitative RT-PCR, Quantitative Proteomics, Standard Deviation

Journal: International Journal of General Medicine

Article Title: microRNA-19a-3p and microRNA-376c-3p Promote Hepatocellular Carcinoma Progression Through SOX6-Mediated Wnt/β-Catenin Signaling Pathway

doi: 10.2147/IJGM.S278538

Figure Lengend Snippet: SOX6 is poorly expressed in HCC tissues and cells. ( A ) SOX6 mRNA expression in 21 cases of HCC tissues and adjacent normal tissues determined by RT-qPCR. ( B ) The positive rate of SOX6 protein expression in 21 cases of HCC tissues and adjacent normal tissues detected by immunohistochemical staining. ( C ) SOX6 mRNA expression in human normal liver cell line THLE3 and HCC cell lines (Hep3B, MHCC-97L, Huh7) determined by RT-qPCR. ( D ) SOX6 protein expression in human normal liver cell line THLE3 and HCC cell lines (Hep3B, MHCC-97L, Huh7) tested by Western blot analysis. ( E ) detection of SOX6 expression in the TCGA database. ( F ) detection of SOX6 expression at different stages of HCC. ( G ) analysis of the prognosis of patients with differential expression of SOX6. ( H ) correlation analysis of miR-19a-3p and miR-376c-3p with SOX6 expression in HCC tissues. The measurement data were depicted as mean ± standard deviation. Comparison between two groups was conducted by unpaired t -test or and comparisons among multiple groups were analyzed by one-way ANOVA. The experiment was repeated three times. * p < 0.05 vs adjacent normal tissues or THLE3 cells.

Article Snippet: Human normal liver cell line THLE3 and HCC cell lines (Hep3B, American Type Culture Collection, Rockville, Maryland, USA; MHCC-97L and Huh7, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., Shanghai, China) were, respectively, seeded in Roswell Park Memorial Institute (RPMI)-1640 cell culture medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Western Blot, Quantitative Proteomics, Standard Deviation, Comparison

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: The THLE3 normal liver cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the immortalized human hepatocyte line MIHA was kindly provided by Dr Roy-Chowdhury (Albert Einstein College of Medicine, Bronx, NY, USA).

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