normal hepatocyte lo2 cell line  (ATCC)

Journal: Translational Cancer Research

Article Title: Analysis of the role of POC1A in the development and progression of hepatocellular carcinoma

doi: 10.21037/tcr-23-2398

Figure Lengend Snippet: Analysis of the expression of POC1A in various classifications of HCC patients, as well as in HCC and liver cancer cell lines. (A) Comparison of POC1A between cancer tissue samples and normal tissue samples. (B) Comparison of the expression of POC1A in different genders. (C) Comparison of the expression of POC1A in different age groups. (D) The expression levels of POC1A were all higher in the Caucasian, African-American, and Asian HCC patients than the normal patients. The expression of POC1A was significantly higher in LIHC tissue samples from Asian patients than LIHC tissue samples from Caucasian patients. (E) The expression of POC1A in individual cancer stages. (F) The expression of POC1A in different transistor grades. (G) The expression of POC1A in tumor (T) stage. (H) The expression of POC1A in node (N) stage. (I) The expression of POC1A in metastasis (M) stage. (J) Analysis of POC1A expression in normal liver tissue and cancer tissue samples from patients with hepatocellular carcinoma using immunohistochemical methods in the Human Protein Atlas database. Liver sample: https://www.proteinatlas.org/ENSG00000164087-POC1A/tissue/liver#img , hepatocellular samples: https://www.proteinatlas.org/ENSG00000164087-POC1A/pathology/liver+cancer#img . Scale bar: 100 µm. (K) Analysis of POC1A mRNA expression in LO2 and HepG2 cell lines by qPCR assay. *, P<0.05; **, P<0.01; -: no significance. TCGA, The Cancer Genome Atlas; HCC, hepatocellular carcinoma; LIHC, liver hepatocellular carcinoma; mRNA, messenger RNA; qPCR, quantitative polymerase chain reaction; POC1A , POC1 centriolar protein A.

Article Snippet: The human normal hepatocyte cell line LO2 (Servicebio Technology Co., Ltd., Wuhan, China) was cultured in Roswell Park Memorial Institute 1640 medium, and the hepatoma cell line HepG2 (Procell Life Science & Technology Co., Ltd., Wuhan, China) was cultured in Dulbecco’s Modified Eagle Medium (Hyclone, Thermo Fisher Scientific Inc., Waltham, MA), supplemented with 10% fetal bovine serum and antibiotics (10,000 U/mL penicillin and 10 mg/mL streptomycin) (Procell Life Science & Technology Co., Ltd.).

Techniques: Expressing, Comparison, Immunohistochemical staining, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: WW domain-binding protein 2 overexpression prevents diet-induced liver steatosis and insulin resistance through AMPKβ1

doi: 10.1038/s41419-021-03536-8

Figure Lengend Snippet: A , C A WBP2-overexpressing cell line ( A ) and a WBP2 knockout cell line ( C ) were established in LO2 cells. The protein expression of WBP2 was tested by western blot analysis. B Representative photomicrographs with Nile red staining are shown for the control and WBP2-overexpressing cells exposed to PA (0.25 mM) for 12 h. D Representative photomicrographs with Nile red staining are shown for the WT and WBP2 KO cells exposed to PA (0.25 mM) for 12 h. The Nile red-stained area was quantified by ImageJ software. Scale bar, 100 μm. Data are expressed as the mean ± SEM, *** P < 0.001.

Article Snippet: The HEK293T and normal human hepatocyte cell line LO2 were provided by Procell Biotech (Wuhan, China).

Techniques: Knock-Out, Expressing, Western Blot, Staining, Control, Software

Journal: Cell Death & Disease

Article Title: WW domain-binding protein 2 overexpression prevents diet-induced liver steatosis and insulin resistance through AMPKβ1

doi: 10.1038/s41419-021-03536-8

Figure Lengend Snippet: A Silver-stained gel of the indicated proteins binding to WBP2, which were coimmunoprecipitated using an anti-WBP2 antibody and identified via mass spectrometry (MS) in LO2 cells. IgG was used as a control. B Immunoprecipitation and western blot assays using anti-WBP2, anti-AMPKα, anti-AMPKβ1, and anti-AMPKγ1 to detect the binding of AMPKα, AMPKβ1, and AMPKγ1 to WBP2 in LO2 cells. IgG was used as a control. C , D GST precipitation assays using anti-GST and anti-Flag to detect the direct binding of WBP2 and AMPKβ1. Purified GST was used as a control. E WBP2 colocalized with AMPKβ1. Immunofluorescence staining for WBP2 (green), AMPKβ1 (red), and DAPI (blue) in LO2 cells. Scale bar, 20 μm. F Western blot assays were performed in liver tissues from the AAV-WBP2 mice and the AAV-control mice fed an HFD for 16 weeks to detect the expression of AMPKβ1, AMPKγ1, AMPKα, p-AMPKα, ACC, and p-ACC. Anti-Flag was used to detect the expression of exogenous WBP2, and β-actin served as the loading control. G Western blot assays were performed in the PA-treated LO2 cells transfected with WBP2 or control to detect the expression of AMPKβ1, AMPKγ1, AMPKα, p-AMPKα, ACC, and p-ACC. β-actin served as loading control. H Western blot assays were performed in liver tissues from the AAV-Scr sh mice and the AAV-WBP2 sh mice fed an HFD for 16 weeks to detect the expression of AMPKβ1, AMPKγ1, AMPKα, p-AMPKα, ACC, and p-ACC. β-actin served as the loading control. I Western blot assays were performed in the PA-treated wild-type (WT) or WBP2-knockout (KO) LO2 cells to detect the expression of AMPKβ1, AMPKγ1, AMPKα, p-AMPKα, ACC, and p-ACC. β-actin served as loading control.

Article Snippet: The HEK293T and normal human hepatocyte cell line LO2 were provided by Procell Biotech (Wuhan, China).

Techniques: Staining, Binding Assay, Mass Spectrometry, Control, Immunoprecipitation, Western Blot, Purification, Immunofluorescence, Expressing, Transfection, Knock-Out

Journal: Cell Death & Disease

Article Title: WW domain-binding protein 2 overexpression prevents diet-induced liver steatosis and insulin resistance through AMPKβ1

doi: 10.1038/s41419-021-03536-8

Figure Lengend Snippet: A Western blot assays using anti-WBP2 and anti-Flag to detect the expression of WBP2 and exogenous AMPKβ1 in WBP2 wild-type (WT) and WBP2 knockout (KO) LO2 cells. β-actin served as loading control. Immunoprecipitation assays using anti-Flag and western blot assays using anti-phospho-(Ser/Thr) (Phos S/T) to detect the phosphorylation of AMPKβ1. B Western blot assays using anti-WBP2 and anti-Flag to detect the expression of WBP2 and exogenous AMPKβ1 (including wild-type (WT) or Ser108 mutated to Ala108 (S108A) plasmids) in the WBP2 knockout (KO) LO2 cells. β-actin served as the loading control. Immunoprecipitation assays using anti-Flag and western blot assays using anti-phospho-(Ser/Thr) (Phos S/T) were used to detect the phosphorylation of AMPKβ1. C Western blot assays were performed to detect the expression of AMPKα, p-AMPKα, ACC, and p-ACC in the WBP2 WT cells with or without WBP2 overexpression, with or without AMPKβ1 siRNA, and with or without AMPKβ1 WT or with the Ser108 to Ala108 mutation (S108A); anti-WBP2 was used to detect the expression of WBP2, and β-actin served as the loading control. Immunoprecipitation assays using anti-Flag and western blot assays using anti-phospho-(Ser/Thr) (Phos S/T) to detect the phosphorylation of AMPKβ1. D Relative AMPK complex activity compared with the wild-type AMPK complex was measured by the ADP-Glo TM kit according to the generation of ADP. Purified AMPK subunits and WBP2 were incubated. Part of the mixture was used to measure luminescence, and western blot assays were used to detect the phosphorylation of AMPKα and AMPKβ. E Representative photomicrographs with Nile red staining are shown for the WBP2 WT cells with or without WBP2 overexpression, with or without AMPKβ1 siRNA, and with or without AMPKβ1 WT or S108A exposed to PA (0.25 mM) for 12 h. The Nile red-stained area was quantified by ImageJ software. Scale bar, 100 μm. Data are expressed as the mean ± SEM, *** P < 0.001 between the two indicated groups.

Article Snippet: The HEK293T and normal human hepatocyte cell line LO2 were provided by Procell Biotech (Wuhan, China).

Techniques: Western Blot, Expressing, Knock-Out, Control, Immunoprecipitation, Phospho-proteomics, Over Expression, Mutagenesis, Activity Assay, Purification, Incubation, Staining, Software

Journal: Cell Death & Disease

Article Title: WW domain-binding protein 2 overexpression prevents diet-induced liver steatosis and insulin resistance through AMPKβ1

doi: 10.1038/s41419-021-03536-8

Figure Lengend Snippet: A LO2 cells were transfected with the WBP2 promoter reporter plasmid and treated with BSA or PA. The luciferase activity was analyzed ( n = 5 per group). B The WBP2-3′-UTR reporter plasmid was transiently transfected into LO2 cells and stimulated as indicated. The luciferase reporter assay was analyzed ( n = 5 per group). C Heat maps showing the changes in the expression of DEGs involved in microRNA upregulation by PA treatment. The color bar shows the gradient of the log2-fold changes in microRNA expression levels in the PA-treated samples relative to those in the BSA-treated samples. D MicroRNAs targeting the 3’-UTR of WBP2 in humans and mice were predicted using multiple databases. A Venn diagram was constructed with the upregulated miRNAs in the heatmap, the intersection was identified, and miR-27a-5p was discovered. E Binding site of miR-27a-5p on the 3’-UTR of WBP2 in human. F The relative expression of miR-27a-5p between BSA- and PA-treated LO2 cells. G The WBP2-3′-UTR reporter plasmid was transfected into LO2 cells with miR-27a-5p antagomir and stimulated with PA or BSA. The luciferase reporter assay was performed ( n = 5 per group). H Wild-type or mutated WBP2 3’-UTR reporters were transfected into LO2 cells with or without miR-27a-5p and stimulated with PA or BSA. The luciferase reporter assay was performed ( n = 5 per group). I Wild-type or mutated WBP2 3’-UTR reporters were transfected into LO2 cells with or without miR-27a-5p and stimulated with PA or BSA. Western blot assays were performed to detect the expression of WBP2, p-AMPKβ1, AMPKβ1, p-AMPKα, AMPKα, and p-ACC and ACC, and β-actin served as a loading control. Data represent the mean ± SEM, ** P < 0.01, *** P < 0.001 and n.s. indicates no significance between the two indicated groups.

Article Snippet: The HEK293T and normal human hepatocyte cell line LO2 were provided by Procell Biotech (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Expressing, Construct, Binding Assay, Western Blot, Control