hep3b cancer cell line  (BioMimetic Therapeutics)

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: Biomimetics

Article Title: Anticancer Activity of Thiophene Carboxamide Derivatives as CA-4 Biomimetics: Synthesis, Biological Potency, 3D Spheroid Model, and Molecular Dynamics Simulation

doi: 10.3390/biomimetics7040247

Figure Lengend Snippet: IC 50 (µM) of phenyl-thiophene-carboxamide compounds ( 2a – 2e ) on several cell lines.

Article Snippet: Our results showed that the PSA of the most synthesized structures was biomimetic to CA-4, and similar chemical and biological properties were observed against Hep3B cancer cell line.

Techniques:

Journal: Biomimetics

Article Title: Anticancer Activity of Thiophene Carboxamide Derivatives as CA-4 Biomimetics: Synthesis, Biological Potency, 3D Spheroid Model, and Molecular Dynamics Simulation

doi: 10.3390/biomimetics7040247

Figure Lengend Snippet: RHA-3 ( 2b ) and RHA-6 ( 2e ) perturb 3D hepatocellular spheroids’ formation. Images of cluster/s formed by Hep3B hepatocellular carcinoma in presence of 2b (17 µg/mL) or 2e (17 µg/mL) after 24 h of treatment compared to controls ( A ). Cluster percentage of occupied area relative to the negative control ( B ), cluster circularity ( C ), and cluster count ( D ). The non-treated cells are referred to as a negative control. At 100 μg/mL, DOX was utilized as a positive control. The scale bar represents a distance of 10 μm. Circularity scale: a value of 1 represents a perfect circle (ns: p > 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, and ****: p ≤ 0.0001).

Article Snippet: Our results showed that the PSA of the most synthesized structures was biomimetic to CA-4, and similar chemical and biological properties were observed against Hep3B cancer cell line.

Techniques: Negative Control, Positive Control

Journal: Molecular Therapy Oncolytics

Article Title: Dual EGFR- and TfR-targeted gene transfer for sodium iodide symporter gene therapy of glioblastoma

doi: 10.1016/j.omto.2022.10.013

Figure Lengend Snippet: Polyplex-mediated NIS gene transfer in vitro Cell surface receptor expression of EGFR and TfR was measured by flow cytometry. A specific antibody monitored the expression levels of human EGFR and TfR on Hep3B, MCF-7, and U87 cells compared with isotype controls (A). 125 I cell transfection studies (n = 3 for each cell line) showed EGFR- and TfR-specific transfection efficiency of targeted polyplexes (GE11/NIS, TfRre/NIS) (B). Dual/NIS polyplexes showed transfection efficiency in Hep3B and U87 cells (B). Background radiation levels after control transfection with LUC-coding polyplexes (Dual/LUC) or the addition of NIS-specific inhibitor perchlorate proved NIS dependency of iodide uptake (B). Treatment with the dynamin inhibitor dynasore resulted in a dose-dependent inhibition of the transfection of MCF-7 and U87 cells using TfRre/NIS polyplexes demonstrating the TfR dependency of transfection with TfR-targeted polyplexes. Total inhibition was reached by the concentration of 40 μM on MCF-7 and 50 μM on U87 cells (∗p ≤ 0.05, ∗∗p ≤ 0.01). Cell viability of Hep3B, MCF-7, and U87 was not affected by polyplex treatment (C). Results are reported as mean ± SEM.

Article Snippet: The human hepatocellular cancer cell line Hep3B (HB-8064; ATCC, Manassas, VA, UDA) was cultured in DMEM (1 g/L glucose; Sigma-Aldrich) supplemented with 1% (v/v) MEM non-essential amino acids (Thermo Fisher Scientific), 1% (v/v) sodium pyruvate (Thermo Fisher Scientific), and 1% (v/v) L-glutamine (Sigma-Aldrich).

Techniques: In Vitro, Cell Surface Receptor Assay, Expressing, Flow Cytometry, Transfection, Control, Inhibition, Concentration Assay