hep3b liver cancer cell line  (Procell Inc)

Journal: Signal Transduction and Targeted Therapy

Article Title: Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

doi: 10.1038/s41392-024-01887-0

Figure Lengend Snippet: Sema3C promotes HCC stemness and initiation. a Sema3C protein expression levels in various HCC cell lines, hepatic stellate cells (LX-2) and normal liver cells (MIHA) were detected by Western blotting. b Sema3C protein expression levels were determined by Western blotting in Sema3C overexpressed and knockdown HCC cells. c Sema3C mRNA expression level in spheroids and non-spheroids cells of HepG2 and Huh7 cells. d The mRNA expression of stemness-associated genes in Sema3C-overexpressing HCC and control cells was determined by qRT-PCR analysis. The effect of Sema3C overexpression ( e ) or knockdown ( f ) on HCC cell chemoresistance was assessed by an MTT assay. The effect of Sema3C overexpression ( g ) or knockdown ( h ) on HCC cells self-renewal was evaluated by sphere formation assay. Scale bar, 100 μm. i Limiting dilution assay was conducted using HepG2 cells with Sema3C knockdown (shSema3C) or Hep3B cells infected with lentivirus-OE-Sema3C or empty vector in BALB/c nude mice (n = 5 per group). Student’s t test was used for comparing two groups and extreme limiting dilution analysis (ELDA) was used for limiting dilution assay. OE overexpression, NC nontarget control. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Liver cancer cell line Hep3B were purchased from Procell (Wuhan, China), liver cancer cell lines (Huh7, MHCC-97L, HepG2 and SMMC7721) and the human HSC line LX-2 were purchased from Immocell (Xiamen, China), Human hepatocytes MIHA cell line was obtained from Fenghui (Hunan, China), all liver cancer cell lines and LX-2 were cultured in DMEM (Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Invitrogen, United States), MIHA cells were culture in RPMI 1640 (Gibco, United States) containing 10% FBS and 1% penicillin-streptomycin.

Techniques: Expressing, Western Blot, Knockdown, Control, Quantitative RT-PCR, Over Expression, MTT Assay, Tube Formation Assay, Limiting Dilution Assay, Infection, Plasmid Preparation

Journal: Signal Transduction and Targeted Therapy

Article Title: Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

doi: 10.1038/s41392-024-01887-0

Figure Lengend Snippet: Sema3C drives HCC initiation via a dysregulated AKT/Gli1/c-Myc signaling axis. a KEGG pathway analysis found Sema3C to be highly correlated with PI3K-AKT signaling in HCC patients based on the TCGA-LIHC database. b GSEA identified an enrichment of genes involved in PI3K/AKT and Hedgehog signaling pathways in the high-Sema3C-expressing HCC group. c Western blotting analyses of the β-catenin and phosphorylated-YAP levels in Sema3C-overexpressing MHCC-97L cells. d The mRNA expression of Gli1, Gli2, c-Myc , and CCND1 in MHCC-97L cells transfected with OE-Sema3C or vector control and HepG2 cells transfected with sh-Sema3C or NC. e The protein expression levels of total AKT, p-AKT, Gli1, and c-Myc in MHCC-97L cells and HepG2 cells. Sema3C-overexpressing Hep3B and MHCC-97L cells were treated with MK2206 (AKT inhibitor) as indicated, and the cell viability was analyzed by an MTT assay ( f ), the ability of self-renewal was determined by a sphere formation assay, scale bar, 100 μm ( g ); the levels of Gli1, p-AKT, AKT, and c-Myc were determined by western blotting ( h ). i H&E and IHC staining for Ki67, c-Myc, p-AKT (Ser473) and Gli1 in HepG2 xenograft tumors. NC nontarget control; H&E hematoxylin-eosin; IHC immunohistochemistry; Data are presented as means ± SD. ns, not significantly; *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Liver cancer cell line Hep3B were purchased from Procell (Wuhan, China), liver cancer cell lines (Huh7, MHCC-97L, HepG2 and SMMC7721) and the human HSC line LX-2 were purchased from Immocell (Xiamen, China), Human hepatocytes MIHA cell line was obtained from Fenghui (Hunan, China), all liver cancer cell lines and LX-2 were cultured in DMEM (Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Invitrogen, United States), MIHA cells were culture in RPMI 1640 (Gibco, United States) containing 10% FBS and 1% penicillin-streptomycin.

Techniques: Protein-Protein interactions, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, MTT Assay, Tube Formation Assay, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

doi: 10.1038/s41392-024-01887-0

Figure Lengend Snippet: Sema3C is involved in the ECM remodeling and induces the HSCs activation. a GO analysis based on intersection proteins binding to Sema3C and NRP1. b Representative images of collagen gel contraction assays with MHCC-97L cells in either MHCC-97L supernatants or OE-Sema3C-MHCC-97L supernatants. Graph quantifying the change in the percentages of gel contraction by MHCC-97L cells in different conditions. Scale bar, 5 mm. c Representative images showing H&E, Masson’s trichrome and picrosirius red staining, and IHC staining for α-SMA and collagen I expression in serial sections of orthotopic liver xenograft tumors of BALB/C nude mice injected with MHCC-97L-Vector or MHCC-97L-OE-Sema3C cells. Gross tissue image scale bar, 1 cm, microscope image scale bar, 200 μm. d Percent of collagen fibers and α-SMA per field were counted in at least three random fields per animal, (n = 5). e Representative immunofluorescence images for α-SMA and collagen I in HSCs treated with dosage rhSema3C or supernatants of different Sema3C expression levels. Scale bar, 50 μm. LX-2 cells were treated with a gradient dose of rhSema3C or supernatants from Sema3C-overexpressed Hep3B and MHCC-97L cells. The α-SMA expression levels were detected by western blotting analysis ( f ). The TGF-β1 secretion was examined by ELISA assay ( g ). The chemoresistance ability was reflected by an MTT assay ( h ). The ability of migration and invasion was performed by Transwell assays, scale bar, 200 μm ( i ). 5 × 10 5 Hep3B cells or OE-Sema3C Hep3B cells were injected subcutaneously into nude mice alone or mixed with LX-2 cells in a 1:1 ratio. The mice were sacrificed, and the xenograft tumors were excised 32 days after inoculation ( j ). The bar chart showed the tumor weight in each treatment group ( k ). The tumor volumes were monitored for 32 days ( l ). n = 5 per group. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Liver cancer cell line Hep3B were purchased from Procell (Wuhan, China), liver cancer cell lines (Huh7, MHCC-97L, HepG2 and SMMC7721) and the human HSC line LX-2 were purchased from Immocell (Xiamen, China), Human hepatocytes MIHA cell line was obtained from Fenghui (Hunan, China), all liver cancer cell lines and LX-2 were cultured in DMEM (Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Invitrogen, United States), MIHA cells were culture in RPMI 1640 (Gibco, United States) containing 10% FBS and 1% penicillin-streptomycin.

Techniques: Activation Assay, Binding Assay, Staining, Immunohistochemistry, Expressing, Injection, Plasmid Preparation, Microscopy, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay, MTT Assay, Migration

Journal: Signal Transduction and Targeted Therapy

Article Title: Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

doi: 10.1038/s41392-024-01887-0

Figure Lengend Snippet: IL-6 and cholesterol biosynthesis are responsible for Sema3C-mediated HSCs activation. a The volcano map showed the differentially expressed genes of LX-2 cells treated with rhSema3C or PBS in vitro. HSCs were treated with rhSema3C or supernatants of MHCC-97L cells with different Sema3C expression levels. mRNA levels of IL6 and IL8 were detected using qRT-PCR ( b ). ELISA was used to examine the IL6 secretion level ( c ). d GSEA identified an enrichment of genes involved in the NF-κB pathway in rhSema3C-treated LX-2 cells. e Western blotting showed phosphorylated and total p65 protein expression levels in LX-2 cells treated with rhSema3C. LX-2 cells were pre-treated with Bay 11-7082 and subsequently stimulated with rhSema3C, the levels of phosphorylated p65, total p65, and α-SMA expression were determined by western blotting ( f ), and IL6 secretion levels were examined by ELISA assay ( g ). h LX-2 cells were treated with OE-Sema3C-Hep3B cell-derived supernatants, Co-IP assay was used to detect the interaction between ITGB1 and NRP1. LX-2 cells were transfected with siNRP1 and subsequently stimulated with rhSema3C, IL6 secretion levels were detected by ELISA ( i ). The phosphorylated p65, total p65, α-SMA, and HMGCR expression levels were determined by using western blotting ( j ). k LX-2 cells were treated with a dosage of rhSema3C or supernatants of Sema3C-overexpressed HCC cells, and ITGB1 protein expression was determined by western blotting. LX-2 cells were transfected with siITGB1 and subsequently stimulated with rhSema3C, and the IL6 secretion levels were detected by ELISA assay ( l ), the phosphorylated p65, total p65, and α-SMA expression levels were determined by using Western blotting ( m ). LX-2 cells were treated with siNRP1, siITGB1 or rhSema3C as indicated, the secretion of TGF-β1 in each group was detected by ELISA ( n ), the chemotherapy resistance of LX-2 cells was evaluated by MTT assay ( o ), and migration and invasion were performed by Transwell assay, scale bar, 200 μm ( p ). rhSema3C recombinant human Sema3C; Data are presented as means ± SD. ns not significantly; *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Liver cancer cell line Hep3B were purchased from Procell (Wuhan, China), liver cancer cell lines (Huh7, MHCC-97L, HepG2 and SMMC7721) and the human HSC line LX-2 were purchased from Immocell (Xiamen, China), Human hepatocytes MIHA cell line was obtained from Fenghui (Hunan, China), all liver cancer cell lines and LX-2 were cultured in DMEM (Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Invitrogen, United States), MIHA cells were culture in RPMI 1640 (Gibco, United States) containing 10% FBS and 1% penicillin-streptomycin.

Techniques: Activation Assay, In Vitro, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Co-Immunoprecipitation Assay, Transfection, MTT Assay, Migration, Transwell Assay, Recombinant

Journal: BMB Reports

Article Title: Melatonin inhibits glycolysis in hepatocellular carcinoma cells by downregulating mitochondrial respiration and mTORC1 activity

doi: 10.5483/BMBRep.2022.55.9.177

Figure Lengend Snippet: Acute effects of melatonin on mitochondrial dysfunction and ROS production in Hep3B and Huh7 cells. (A) Mitochondrial oxygen consumption rate (OCR) in Hep3B and Huh7 cells treated with or without melatonin (2 mM) at the indicated times following exposure to oligomycin (1 μM), CCCP (5 μM), rotenone (1 μM). Data are expressed as ratios relative to baseline, i.e., Relative basal OCR (B), relative maximal respiration (C), and relative ATP-linked respiration (D) in the melatonin-treated HCC cells shown in (A). (E) Relative glucose uptake in Hep3B and Huh7 cells treated with or without melatonin for 10 min. (F) Extracellular acidification rate (ECAR) in Hep3B and Huh7 cells treated with or without melatonin (2 mM) at the indicated times with glucose (10 mM), oligomycin (1 μM), and 2-deoxyglucose (2-DG; 100 mM). Data are expressed as ratios relative to the baseline. (G) Relative glycolysis in the HCC cells shown in (F). Data are expressed as the mean ± SEM (n = 4-5). (H) Representative fluorescence micrographs showing mitochondrial superoxide production by Hep3B and Huh7 cells treated with or without melatonin for 30 min. (I) Bar graph showing quantitation of MitoSOX fluorescence intensity. Scale bar, 20 μM. Data are expressed as the mean ± SEM (n = 3). n.s. denotes ‘not significant’; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The liver cancer cell lines Hep3B (ATCC, Manassas, VA, USA) and Huh7 (Korean Cell Line Bank, Seoul, Korea) were cultured in EMEM and DMEM medium, respectively, containing 10% fetal bovine serum and 1% penicillin/streptomycin (P/S).

Techniques: Fluorescence, Quantitation Assay

Journal: BMB Reports

Article Title: Melatonin inhibits glycolysis in hepatocellular carcinoma cells by downregulating mitochondrial respiration and mTORC1 activity

doi: 10.5483/BMBRep.2022.55.9.177

Figure Lengend Snippet: Temporal effects of melatonin on mTORC1 and glycolytic proteins in Hep3B and Huh7 cells. (A, B) Levels of phosphorylated mTOR, S6K, c-Myc, LDHA, and HK2 protein in Hep3B and Huh7 cells treated with or without melatonin for (A) 3 h or (B) 48 h. (C, D) Levels of c-Myc, LDHA, and HK2 mRNA in Hep3B and Huh7 cells treated with or without melatonin for (C) 3 h or (D) 48 h. Data are normalized against 36B4 mRNA levels and expressed as the mean ± SEM of three independent experiments. (E, F) Effects of c-Myc on levels of LDHA and HK2 in (E) Hep3B and (F) Huh7 cells treated with melatonin (2 mM) for 48 h. n.s., denotes ‘not significant’; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The liver cancer cell lines Hep3B (ATCC, Manassas, VA, USA) and Huh7 (Korean Cell Line Bank, Seoul, Korea) were cultured in EMEM and DMEM medium, respectively, containing 10% fetal bovine serum and 1% penicillin/streptomycin (P/S).

Techniques:

Journal: BMB Reports

Article Title: Melatonin inhibits glycolysis in hepatocellular carcinoma cells by downregulating mitochondrial respiration and mTORC1 activity

doi: 10.5483/BMBRep.2022.55.9.177

Figure Lengend Snippet: Chronic effects of melatonin on mitochondrial dysfunction and glycolysis in Hep3B and Huh7 cells. (A) Mitochondrial oxygen consumption rate and (B) extracellular acidification rate in Hep3B and Huh7 cells treated with or without melatonin (2 mM) for 48 h following exposure to oligomycin (1 μM), CCCP (5 μM), rotenone (1 μM), glucose (10 mM), and 2-deoxyglucose (2-DG; 100 mM). (C) Basal OCR, (D) maximal respiration, and (E) ATP-linked respiration in the Hep3B and Huh7 cells shown in (A). (F) Glycolysis in the Hep3B and Huh7 cells shown in (B). Data are expressed as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The liver cancer cell lines Hep3B (ATCC, Manassas, VA, USA) and Huh7 (Korean Cell Line Bank, Seoul, Korea) were cultured in EMEM and DMEM medium, respectively, containing 10% fetal bovine serum and 1% penicillin/streptomycin (P/S).

Techniques:

Journal: BMB Reports

Article Title: Melatonin inhibits glycolysis in hepatocellular carcinoma cells by downregulating mitochondrial respiration and mTORC1 activity

doi: 10.5483/BMBRep.2022.55.9.177

Figure Lengend Snippet: Melatonin inhibits Hep3B and Huh7 cell proliferation and increases apoptosis. (A-C) Hep3B and Huh7 cells were treated with or without melatonin for 48 h. (A) Relative cell number and (B, C) measurement of ATP- or NADH&NADPH-based cell viability in melatonin-treated Hep3B and Huh7 cells. (D) Effects of c-Myc (as shown in ) on relative cell numbers of Hep3B and Huh7 cells treated with melatonin (2 mM) for 48 h. (E) Clonogenic assay of Hep3B and Huh7 cells treated with or without melatonin for 10 days (left panel). Quantification of the number of colonies following treatment with the indicated concentrations of melatonin (right panel). (F) Relative caspase 3/7 activity in Hep3B and Huh7 cells treated with melatonin for 48 h. (G) Levels of cleaved caspase-9, -3, -7 and PARP-1 in Hep3B and Huh7 cells treated with melatonin for 48 h. (H) Schematic showing the role of melatonin in inhibition of glycolysis via downregulation of mitochondrial respiration and mTORC1 activity. Data are expressed as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The liver cancer cell lines Hep3B (ATCC, Manassas, VA, USA) and Huh7 (Korean Cell Line Bank, Seoul, Korea) were cultured in EMEM and DMEM medium, respectively, containing 10% fetal bovine serum and 1% penicillin/streptomycin (P/S).

Techniques: Clonogenic Assay, Activity Assay, Inhibition