thle-2  (ATCC)

Journal: International Journal of Biological Sciences

Article Title: Kupffer Cell-derived IL6 Promotes Hepatocellular Carcinoma Metastasis Via the JAK1-ACAP4 Pathway

doi: 10.7150/ijbs.97109

Figure Lengend Snippet: IL6 promotes cancer cell migration through the ACAP4-ARF6 pathway. (A, B) Statistical analysis of the migration of MHCC97H and HepG2 cells after IL6 stimulation utilizing wound healing assay. The data are presented as the average ± SEM. * p < 0.05, ** p < 0.01. (C, D) Quantitative analysis of the fluorescence intensity of ACAP4-FITC and ARF6-mCherry in the control and IL6-treated groups corresponding to the dashed line in the fluorescence images of MHCC97H cells. (E) MHCC97H cells were transfected with ARF6-mCherry plasmids for 24 h and then stimulated with IL6 for 20 min. The cells were fixed and stained with an anti-ACAP4 antibody. The images were acquired under a Delta Vision microscope (scale bar, 10 μm). (F, G) Quantitative analysis of the fluorescence intensity of ACAP4-FITC and ARF6-mCherry in the control and IL6-treated groups corresponding to the dashed line in the fluorescence images of HepG2 cells. (H) HepG2 cells were transfected with ARF6-mCherry plasmids for 24 h and then stimulated with IL6 for 20 min. The cells were fixed and stained with an anti-ACAP4 antibody. The images were acquired under a Delta Vision microscope (scale bar, 10 μm). (I, J) Statistical analysis of the ARF6 protein levels in both cytoplasmic and membrane fractions, under conditions with and without IL6 stimulation in MHCC97H and HepG2 cells. (K, L) Statistical analysis of the wound healing assay results for MHCC97H and HepG2 cells and ACAP4 KO (ACAP4 KO ) MHCC97H and HepG2 cells stimulated with IL6 (50 ng/mL) for 12 h. The data are presented as the average ± SEM. ** p < 0.01. (M, N) Statistical analysis of the transwell migration assay results for MHCC97H and HepG2 cells and ACAP4 KO MHCC97H and HepG2 cells stimulated with IL6 (50 ng/mL) for 24 h. The data are presented as the average ± SEM. ** p < 0.01.

Article Snippet: MHCC97H, HepG2, THLE-2, and 293T cells were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Migration, Wound Healing Assay, Fluorescence, Control, Transfection, Staining, Microscopy, Membrane, Transwell Migration Assay

Journal: International Journal of Biological Sciences

Article Title: Kupffer Cell-derived IL6 Promotes Hepatocellular Carcinoma Metastasis Via the JAK1-ACAP4 Pathway

doi: 10.7150/ijbs.97109

Figure Lengend Snippet: IL6-induced ACAP4 phosphorylation promotes ARF6-GTPase activity and cancer cell migration. (A) Endogenous ARF6-GTPase activity was measured by a GGA3 pull-down assay with IL6 simulation. 293T cells coexpressing Flag-ARF6 and GFP-ACAP4 WT were incubated with GST-GGA3 after IL6 stimulation. The active forms of ARF6 were measured with an anti-ARF6 antibody. (B) Quantitative analyses of ARF6-GTPase activity are described in ( A ). The data are presented as the average ± SEM. ** p < 0.01. (C) ARF6-GTPase activity was measured via a GGA3 pull-down assay with different ACAP4 mutants. 293T cells that coexpressed Flag-ARF6 and GFP-ACAP4 WT , GFP-ACAP4 Y843E , or GFP-ACAP4 Y843F were incubated with GST-GGA3. The active forms of ARF6 were measured with an anti-ARF6 antibody. (D) Quantitative analyses of ARF6-GTPase activity are described in (C). The data are presented as the average ± SEM. ** p < 0.01. (E) Flowchart of the construction of the ACAP4 KO cell line and stable expression of the exogenous ACAP4 WT , ACAP4 Y843E , and ACAP4 Y843F cell lines in hepatoma cells. (F, G) ACAP4 KO MHCC97H and HepG2 cells were infected with lentivirus and expressed ACAP4 WT , ACAP4 Y843E , or ACAP4 Y843F ; then, the expression of exogenous ACAP4 WT , ACAP4 Y843E , or ACAP4 Y843F was assessed by IB. (H, I) Quantitative analysis of wound healing in ACAP4 KO MHCC97H and HepG2 cells expressing ACAP4 WT , ACAP4 Y843E , or ACAP4 Y843F , with or without IL6 stimulation. (J, K) Quantitative analysis of transwell migration assay in ACAP4 KO MHCC97H and HepG2 cells expressing ACAP4 WT , ACAP4 Y843E , or ACAP4 Y843F , with or without IL6 stimulation. The data are presented as the average ± SEM. * p < 0.05, ** p < 0.01, ns p > 0.05.

Article Snippet: MHCC97H, HepG2, THLE-2, and 293T cells were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Activity Assay, Migration, Pull Down Assay, Incubation, Expressing, Infection, Transwell Migration Assay

Journal: International Journal of Biological Sciences

Article Title: Kupffer Cell-derived IL6 Promotes Hepatocellular Carcinoma Metastasis Via the JAK1-ACAP4 Pathway

doi: 10.7150/ijbs.97109

Figure Lengend Snippet: ACAP4 Y843 is phosphorylated in HCC tissues and may be a biomarker for predicting HCC metastasis. (A, C) IB analyses of endogenous ACAP4 Y843 /ACAP4 and p-JAK1/JAK1 ratios in MHCC97H and HepG2 cells after IL6 stimulation. (B, D) Quantitative analyses of the ACAP4 Y843 /ACAP4 ratio are described in ( A, C ). The data are presented as the average ± SEM. * p < 0.05, ** p < 0.01. (E) MHCC97H cells were transfected with Flag-ACAP4 or Flag-ACAP4 Y843F for IP after IL6 stimulation, and the tyrosine phosphorylation level of ACAP4 Y843 was analyzed by IB with an anti-p-ACAP4 Y843 antibody. (F) Quantitative analyses of the ACAP4 Y843 /ACAP4 ratio described in ( E ). The data are presented as the average ± SEM. * p < 0.05, ns p > 0.05. (G, H) MHCC97H and HepG2 cells were starved of serum for 4 h before being stimulated with IL6 for 20 min. Then, the cells were fixed, permeabilized, and stained with the p-ACAP4 Y843 antibody. The images were acquired under a Delta Vision microscope (scale bar, 10 μm). (I, J) Statistical analysis of the expression levels of ACAP4, p-ACAP4 Y843 , JAK1 and p-JAK1 are described in ( K ). The data are presented as the average ± SEM. ** p < 0.01, ns p > 0.05. (K) HE staining and expression levels of ACAP4, p-ACAP4 Y843 , JAK1, and p-JAK1 in tumor tissues and adjacent tissues from representative patients with HCC were detected by immunohistochemistry (scale bar, 100 μm).

Article Snippet: MHCC97H, HepG2, THLE-2, and 293T cells were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Biomarker Assay, Transfection, Staining, Microscopy, Expressing, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Kupffer Cell-derived IL6 Promotes Hepatocellular Carcinoma Metastasis Via the JAK1-ACAP4 Pathway

doi: 10.7150/ijbs.97109

Figure Lengend Snippet: Bufalin inhibits cell migration and tumor metastasis in HCC cells and orthotopic xenograft mice. (A, B) Quantitative analyses were conducted on the wound healing assay involving MHCC97H and HepG2 cells that were pretreated with bufalin or itacitinib, followed by stimulation with IL6 at a concentration of 50 ng/mL for a duration of 12 h. The data are presented as the average ± SEM. ** p < 0.01, ns p > 0.05. (C, D) Quantitative analyses were conducted on the transwell migration assay involving MHCC97H and HepG2 cells that were pretreated with bufalin or itacitinib, followed by stimulation with IL6 at a concentration of 50 ng/mL. The data are presented as the average ± SEM. ** p < 0.01, ns p > 0.05. (E) Bioluminescence images of MHCC97H-luciferase cell orthotopic xenograft mice. (F) Quantification of bioluminescent signals (photons/sec) from HCC orthotopic xenograft mice. The data are presented as the average ± SEM. n=5. * p < 0.05. (G) The expression levels of ACAP4, p-ACAP4 Y843 , JAK1, and p-JAK1 in tumor tissues from representative HCC orthotopic xenograft mice were detected by immunohistochemistry (scale bar, 100 μm). (H) Statistical analysis of the expression levels of p-ACAP4 Y843 and p-JAK1 in tumor tissues from orthotopic xenograft mice. The data are presented as the average ± SEM. ** p < 0.01. (I) The expression levels of ACAP4, p-ACAP4 Y843 , JAK1, and p-JAK1 in tumor tissues from representative HCC orthotopic xenograft mice were detected by IB. (J) Statistical analysis of the expression levels of p-ACAP4 Y843 and p-JAK1 in tumor tissues from orthotopic xenograft mice. The data are presented as the average ± SEM. * p < 0.05, ** p < 0.01. (K) The model illustrates the proposed role of IL6 from HCC-educated KCs in regulating HCC metastasis via the JAK1‒ACAP4 pathway.

Article Snippet: MHCC97H, HepG2, THLE-2, and 293T cells were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Migration, Wound Healing Assay, Concentration Assay, Transwell Migration Assay, Luciferase, Expressing, Immunohistochemistry

Journal: Experimental & Molecular Medicine

Article Title: VIM-AS1 , which is regulated by CpG methylation, cooperates with IGF2BP1 to inhibit tumor aggressiveness via EPHA3 degradation in hepatocellular carcinoma

doi: 10.1038/s12276-024-01352-6

Figure Lengend Snippet: a Schematic illustration showing the identification of 50 hypermethylated sites in HCC on the basis of methylome data derived from the CGRC and TCGA (left). Fifty hypermethylated sites were located on CpG islands, shores, shelves, and the open sea (middle). Distribution of the distance between the 50 hypermethylated sites and the TSS (right). b Analysis of the beta values for cg02746869 in multiple malignancies. c Heatmap representing the pattern of VIM-AS1 expression and cg02746869 methylation in various types of cancer (left). Correlation analysis of cg02746869 beta values and VIM-AS1 expression levels (right). d Methylation of cg02746869 and associated VIM-AS1 expression in THLE2 and HUH1. e Increased methylation levels were confirmed using HRM and BS-seq, and decreased VIM-AS1 expression was detected in the DNMT3A experimental set. A decrease in methylation was confirmed using HRM and BS-seq, and VIM-AS1 expression was verified in the TET1 experimental set. f Genomic viewer showing enrichment of active markers at cg02746869 in THLE2 and HUH1. g ChIP‒qPCR for assessing histone activity and TF binding following the methylation and demethylation of cg02746869. Con = empty vector, DNMT3A = hypermethylated cg02746869, TET1 = hypomethylated cg02746869. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human liver epithelial cells (THLE2; CRL-2706 TM ) were purchased from American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Expressing, Methylation, Activity Assay, Binding Assay, Plasmid Preparation

Journal: Experimental & Molecular Medicine

Article Title: VIM-AS1 , which is regulated by CpG methylation, cooperates with IGF2BP1 to inhibit tumor aggressiveness via EPHA3 degradation in hepatocellular carcinoma

doi: 10.1038/s12276-024-01352-6

Figure Lengend Snippet: a Representative image of VIM-AS1 localization was detected using FISH (left). Relative intensity of VIM-AS1 , indicating the percentage of subcellular localization (middle). Comparison of the intensity of each subcellular localization between THLE2 and HUH1 (right). b Validation of VIM-AS1 -overexpressing cells via qRT‒PCR (left) and RNA-seq (right). c DEG correlation profile of up- and downregulated genes after overexpressing VIM-AS1 (left). Red = top 5 upregulated genes; blue = top 5 downregulated genes. GO analysis of DEGs downregulated by VIM-AS1 (right). d GSEA results of downregulated DEGs associated with cell adhesion. e qRT‒PCR of cell adhesion-associated genes in VIM-AS1 -overexpressing cells. f Representative images and quantification of migration and invasion assays. Con = Control, VIM-AS1 OE = VIM-AS1 overexpression, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Human liver epithelial cells (THLE2; CRL-2706 TM ) were purchased from American Type Culture Collection (ATCC).

Techniques: Comparison, RNA Sequencing Assay, Migration, Control, Over Expression

Journal: Experimental & Molecular Medicine

Article Title: VIM-AS1 , which is regulated by CpG methylation, cooperates with IGF2BP1 to inhibit tumor aggressiveness via EPHA3 degradation in hepatocellular carcinoma

doi: 10.1038/s12276-024-01352-6

Figure Lengend Snippet: a Relative mRNA expression of EPHA3 in THLE2, HUH1 and samples with modified methylation. Changes in EPHA3 mRNA expression are shown in response to the modulation of VIM-AS1 . VIM-AS1 OE = VIM-AS1 overexpression, VIM-AS1 KD = VIM-AS1 knockdown. b RNA stability assay for EPHA3 mRNA after actinomycin D treatment of HUH1 cells. Con = Control, OE = VIM-AS1 overexpression. c Immunoblot of EPHA3 protein after VIM-AS1 overexpression. d Cell migration and invasion assays following EPHA3 mRNA knockdown. *p < 0.05, **p < 0.01, ***p < 0.001. Con = Control, EPHA3 KD = EPHA3 knockdown. e VIM-AS1 and EPHA3 RNA expression according to the stage was analyzed using GEPIA. f Probability of survival according to EPHA3 mRNA in all patients with HCC and with MVI analyzed using Kaplan–Meier plotter.

Article Snippet: Human liver epithelial cells (THLE2; CRL-2706 TM ) were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Modification, Methylation, Over Expression, Knockdown, Stability Assay, Control, Western Blot, Migration, RNA Expression

Journal: Experimental & Molecular Medicine

Article Title: VIM-AS1 , which is regulated by CpG methylation, cooperates with IGF2BP1 to inhibit tumor aggressiveness via EPHA3 degradation in hepatocellular carcinoma

doi: 10.1038/s12276-024-01352-6

Figure Lengend Snippet: RIP-qPCR assay showing the binding affinity of VIM-AS1 and EPHA3 mRNAs for IGF2BP1 following the overexpression of VIM-AS1 in HUH1 cells ( a ) and VIM-AS1 knockdown in THLE2 cells ( b ). c A schematic illustrating how the effect of increasing/decreasing VIM-AS1 expression on EPHA3 mRNA is mediated by IGF2BP1. d FISH results showing the localization of VIM-AS1 and IGF2BP1 (left) and EPHA3 mRNA and IGF2BP1 (right). Bar graphs showing the relative colocalization fluorescence intensity of each FISH dataset. White arrows = colocalization region of VIM-AS1 or EPHA3 mRNA and IGF2BP1. Con Control, OE = VIM-AS1 overexpression, KD = VIM-AS1 knockdown, ND not detected, ***p < 0.001.

Article Snippet: Human liver epithelial cells (THLE2; CRL-2706 TM ) were purchased from American Type Culture Collection (ATCC).

Techniques: Binding Assay, Over Expression, Knockdown, Expressing, Fluorescence, Control