mouse hepatoma cell line hepa1  (DSMZ)

Journal: Journal of Extracellular Vesicles

Article Title: A Unique Intercellular Feedforward Loop From HK1 to TGF‐β1 Promotes the Progression of Hepatocellular Carcinoma

doi: 10.1002/jev2.70255

Figure Lengend Snippet: Glucose metabolism of HCC cells affects their ability of activating HSC. (A) The human HCC scRNA‐seq ( n = 29 patients, GSE151530 ) was analysed to demonstrate the correlation between the glucose metabolism (reactome‐glucose metabolism) in HCC cells and the ACTA2 expression in HSCs. (B) LX‐2 cells were cultured in the presence of conditional medium from Huh7 cells or HK1‐overexpressing Huh7 cells for 1 week. Cell morphology and α‐SMA expression of LX‐2 cells were indicated. (C) The supernatants of the Huh7 cells treated with or without 2‐DG (5 mM) were collected to incubate LX‐2 cells for 1 week. α‐SMA expression of LX‐2 cells was indicated. (D) Primary HSCs from C57BL/6 mice were incubated with the medium of Hepa1‐6 cells or HK1‐overexpressing Hepa1‐6 cells, α‐SMA expression of primary HSCs was indicated. (E) Hepa1‐6 cells were treated with or without 2‐DG (5 mM) for 24 h, and then the corresponding supernatants were collected to incubate primary HSCs. (F, G) LX‐2 cells were initially treated with TGF‐β1 (2 ng/mL) for 48 h, and then lEVs were collected. Nanoflow cytometry was applied to characterise the size profile (F) and number (G) of isolated lEVs ( n = 3 independent experiments). (H) Transmission electron microscopy was applied to visualise the isolated lEVs. (I) HK1 protein was detected in lEVs‐derived from indicated LX‐2 cells. Flotillin‐2 was used as a loading control for lEVs. WCL, whole cell lysates. (J) lEVs derived from activated control and HK1‐knockdown LX‐2 cells were collected to incubate Huh7 cells. HK1 protein level in Huh7 cells was detected. (K) As the diagram shown, LX‐2 cells were incubated with the corresponding supernatants of indicated Huh7 cells, then α‐SMA expression of LX‐2 cells was indicated. (L) LX‐2 cells were cultured with normal DMEM or medium of Huh7 cells or HK1‐overexpressing Huh7 cells for 1 week, then refresh normal DMEM for culturing another 48 h to collect their lEVs. (M) The correlation between HK1 and ACTA2 expression in HSCs were analysed using the human HCC scRNA‐seq data ( GSE151530 ). (N) The expression levels of ACTA2 in HSCs were analysed using human HCC [ n (low) = 14 tissues, n (high) = 15 tissues, GSE151530 ] and normal liver [ n (low) = 4 tissues, n (high) = 4 tissues, GSE158723 ] scRNA‐seq data. Statistical data are presented as mean ± s.e.m. of indicated samples. Statistical analyses were determined by Pearson correlation (A, M), unpaired two‐tailed Student's t test (G) and Wilcoxon test (N). All western blots were repeated at least twice, and one of them is shown. α‐SMA, alpha smooth muscle actin; 2‐DG, 2‐deoxy‐D‐glucose; DMEM, Dulbecco's modified Eagle's medium; HCC, hepatocellular carcinoma; HK1, hexokinase 1; HSC, hepatic stellate cell; lEV, large extracellular vesicle; TGF‐β1, transforming growth factor‐β1.

Article Snippet: The human embryonic kidney cell line HEK293T (CRL‐11268), human hepatoma cell line HepG2 (HB‐8065) and mouse hepatoma cell line Hepa1‐6 (CRL‐1830) were obtained from American Type Culture Collection.

Techniques: Expressing, Cell Culture, Incubation, Cytometry, Isolation, Transmission Assay, Electron Microscopy, Derivative Assay, Control, Knockdown, Two Tailed Test, Western Blot, Modification

Journal: Journal of Extracellular Vesicles

Article Title: A Unique Intercellular Feedforward Loop From HK1 to TGF‐β1 Promotes the Progression of Hepatocellular Carcinoma

doi: 10.1002/jev2.70255

Figure Lengend Snippet: HK1 delivered from HSCs enhances the capacity of HCC cells to activate HSC. (A) The activity of TGF‐β signalling pathway (Gene Ontology biological processes‐response to TGF‐β) in HK1‐low and HK1‐high HSCs were demonstrated by analysing human HCC ( GSE151530 ) and normal liver ( GSE158723 ) scRNA‐seq data. (B) TGF‐β1 mRNA levels in distinct cell types were analysed with clinical HCC scRNA‐seq (PRJCA007744). (C) Overexpression of Flag‐HK1 in Huh7 cells was conducted, followed by the detection of HK1 expression levels and TGF‐β1 secretion from these cells ( n = 3 independent experiments). (D) TGF‐β1 secretion is detected in Hepa 1–6 cells, HK1‐overexpressing Hepa1‐6 cells and tumour‐associated macrophages ( n = 3 independent experiments). (E, F) Overexpressing HK1 in Huh7 cells and LX‐2 cells were cocultured in a transwell‐based system with distinct Huh7 cells, with or without the addition of TGF‐β1 neutralising antibody. α‐SMA expression and Smad3 phosphorylation of LX‐2 cells were detected. (G) Control and HK1‐knockdown LX‐2 cells were initially treated with TGF‐β1 for 48 h, then lEVs derived from LX‐2 cells were collected to incubate Huh7 cells. The HK1 expression in Huh7 cells and the secretion of TGF‐β1 from Huh7 cells were indicated ( n = 3 independent experiments). (H) Overexpression of Flag‐HK1 or Flag‐HK1 6CS in LX‐2 cells was conducted, the cells were then treated with TGF‐β1 to obtain distinct lEVs. Corresponding lEVs were applied to incubate Huh7 cells, and the HK1 expression and the TGF‐β1 secretion of the Huh7 cells were detected ( n = 3 independent experiments). (I) HK1 and HK2 protein levels were detected in distinct HCC cell lines under both basal and lEV incubation conditions. (J) Control and HK2‐knockdown Huh7 cells were incubated with lEVs derived from activated LX‐2 cells. HKs expression and TGF‐β1 secretion ( n = 3 independent experiments) were assessed. Statistical data are presented as mean ± s.e.m. of indicated samples. Statistical analyses were determined by the Wilcoxon test (A), unpaired two‐tailed Student's t test (C), one‐way analysis of variance (ANOVA) with Tukey's multiple comparisons test (D, G, H) and two‐way ANOVA with Tukey's multiple comparisons test (J). All western blots were repeated at least twice, and one of them is shown. α‐SMA, alpha smooth muscle actin; HCC, hepatocellular carcinoma; HK1, hexokinase 1; HSC, hepatic stellate cell; lEV, large extracellular vesicle; TGF‐β, transforming growth factor‐β.

Article Snippet: The human embryonic kidney cell line HEK293T (CRL‐11268), human hepatoma cell line HepG2 (HB‐8065) and mouse hepatoma cell line Hepa1‐6 (CRL‐1830) were obtained from American Type Culture Collection.

Techniques: Activity Assay, Over Expression, Expressing, Phospho-proteomics, Control, Knockdown, Derivative Assay, Incubation, Two Tailed Test, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: A Unique Intercellular Feedforward Loop From HK1 to TGF‐β1 Promotes the Progression of Hepatocellular Carcinoma

doi: 10.1002/jev2.70255

Figure Lengend Snippet: Intercellular communication from HK1 to TGF‐β1 promotes HCC progression. (A–D) TGF‐β1 WT‐ and TGF‐β1 3NQ‐expressing Hepa1‐6 were orthotopically implanted into the livers of control ( Hk f/f ) and HK1 CKO ( Hk f/f ; Gfap‐Cre ) mice, and representative image and weight of the Hepa1‐6‐derived xenografts were shown (A, n = 8 mice). Expression of HK1, α‐SMA and Ki67 (B, n = 12 fields from three independent mice), Smad3 phosphorylation (C, the p‐Smad3 signal in the α‐SMA‐positive cells was quantified, n = 3 mice) and TGF‐β1 N‐glycosylation (D) from the corresponding tumour samples were indicated. (E–H) TGF‐β1‐ and TGF‐β1 3NQ‐expressing Hepa1‐6 were orthotopically implanted into the livers of control ( Hk f/f ) and HK1 CKO ( Hk f/f ; Lrat‐Cre ) mice. Tumour image and weight ( n = 8 mice), HK1, α‐SMA, Ki67 ( n = 12 fields from three independent mice) and Smad3 phosphorylation expression ( n = 3 mice) and TGF‐β1 N‐glycosylation were detected ( n = 3 mice). The p‐Smad3 signal in the α‐SMA‐positive cells was quantified. (I–K) Representative image and weight of the Hepa1‐6‐derived xenografts in control ( Gfap‐Cre ) C57BL/6 mice and transgenic mice with conditional overexpression of HK1 ( LSL‐HK1; Gfap‐Cre ) or HK1 6CS ( LSL‐HK1 6CS; Gfap‐Cre ) in HSCs (I, n = 8 mice). Expression of HK1, α‐SMA and Ki67 ( n = 12 fields from three independent mice) and Smad3 phosphorylation ( n = 3 mice) from the corresponding tumour samples were indicated. The p‐Smad3 signal in the α‐SMA‐positive cells was quantified. Statistical data are presented as mean ± s.e.m. of indicated samples. Statistical analyses were determined by two‐way ANOVA with Tukey's multiple comparisons test (A–C, E–G) and one‐way ANOVA with Tukey's multiple comparisons test (I–K). All western blots were repeated at least twice, and one of them is shown. α‐SMA, alpha smooth muscle actin; ANOVA, analysis of variance; HK1, hexokinase 1; TGF‐β, transforming growth factor‐β.

Article Snippet: The human embryonic kidney cell line HEK293T (CRL‐11268), human hepatoma cell line HepG2 (HB‐8065) and mouse hepatoma cell line Hepa1‐6 (CRL‐1830) were obtained from American Type Culture Collection.

Techniques: Expressing, Control, Derivative Assay, Phospho-proteomics, Glycoproteomics, Transgenic Assay, Over Expression, Western Blot