Journal: Cancer Management and Research

Article Title: microRNA-145-5p Inhibits Migration, Invasion, and Metastasis in Hepatocellular Carcinoma by Inhibiting ARF6

doi: 10.2147/cmar.s300678

Figure Lengend Snippet: Figure 3 ARF6 was proven to be a novel target of miR-145-5p in HCCLM3 cells. (A) ARF6 had a predicted consequential pairing with miR-145-5p at nucleotide positions 2171–2177. psiCHECK2 dual-luciferase reporter vectors containing WT or MT ARF6 3ʹ-UTR were constructed. (B) Renilla luciferase activity was normalized to firefly luciferase activity in HCCLM3 cells expressing WT-ARF6 or MT-ARF6. (C) After transfection with miR-145 mimics, the ARF6 levels were significantly decreased in HCCLM3 cells. (D) RT-qPCR was performed to measure the expression level of ARF6 in THLE-2 cells. **P<0.01 compared with the 3ʹ-UTR+miR-145 mimics group and ***P<0.001 compared with the NC group. The data are presented as the mean ± standard deviation values. Abbreviations: ARF6, ADP-ribosylation factor 6; NC, negative control; UTR, 3ʹ untranslated regions; WT, wild-type; MT, mutated.

Article Snippet: ARF6 lentiviral activation particles (sc-400799-LAC), which could specifically and efficiently upregulate gene expression when introduced into cells via lentiviral transduction, and control lentiviral activation particles (sc-437282) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Luciferase, Construct, Activity Assay, Expressing, Transfection, Quantitative RT-PCR, Standard Deviation, Negative Control

Journal: Cancer Management and Research

Article Title: microRNA-145-5p Inhibits Migration, Invasion, and Metastasis in Hepatocellular Carcinoma by Inhibiting ARF6

doi: 10.2147/cmar.s300678

Figure Lengend Snippet: Figure 4 Overexpression of ARF6 counteracted the effects of miR-145-5p in HCCLM3 cells. (A) The mRNA level of ARF6 increased after transfection of ARF6 lentiviral activation particles. ***P<0.001 compared with the control lentiviral activation particles. (B) ARF6 levels were measured by RT-qPCR in HCCLM3 cells. (C) Migration rates of HCCLM3 cells were confirmed by a wound healing assay. (D) The invasive ability of transfected HCCLM3 cells was measured by Transwell assay (magnification, 200×). (E) Apoptosis induced by miR-145 was reversed by ARF6. (F) The effects of miR-145 and ARF6 on the cell division cycle were measured by cell cycle analysis. **P<0.01 and ***P<0.001 compared with the miR-145 mimics group. The data are presented as the mean ± standard deviation values. Abbreviations: ARF6, ADP-ribosylation factor 6; NC, negative control.

Article Snippet: ARF6 lentiviral activation particles (sc-400799-LAC), which could specifically and efficiently upregulate gene expression when introduced into cells via lentiviral transduction, and control lentiviral activation particles (sc-437282) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Over Expression, Transfection, Activation Assay, Control, Quantitative RT-PCR, Migration, Wound Healing Assay, Transwell Assay, Cell Cycle Assay, Standard Deviation, Negative Control

Journal: Cancer Management and Research

Article Title: microRNA-145-5p Inhibits Migration, Invasion, and Metastasis in Hepatocellular Carcinoma by Inhibiting ARF6

doi: 10.2147/cmar.s300678

Figure Lengend Snippet: Figure 5 ARF6 was significantly upregulated in HCC tissues and negatively correlated with miR-145 in HCC. (A) Real-time PCR was performed to measure ARF6 mRNA levels in HCC tissues and their matched ANLTs (n=75). ***P<0.001 compared with ANLTs. (B) In HCC tissues, ARF6 levels were negatively correlated with miR-145-5p levels (n=75). (C) ARF6 mRNA levels were measured in HCC cell lines and THLE-2 cells. *P<0.05 compared with THLE-2 cells. (D) Representative immunohistochemical images of ARF6 expression in HCC tissues with different miR-145 levels. ***P<0.001 compared between the two groups. The data are presented as the mean ± standard deviation values. Abbreviations: HCC, hepatocellular carcinoma; ARF6, ADP-ribosylation factor 6.

Article Snippet: ARF6 lentiviral activation particles (sc-400799-LAC), which could specifically and efficiently upregulate gene expression when introduced into cells via lentiviral transduction, and control lentiviral activation particles (sc-437282) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hepatitis B Virus Utilizes a Retrograde Trafficking Route via the Trans-Golgi Network to Avoid Lysosomal Degradation

doi: 10.1016/j.jcmgh.2022.10.008

Figure Lengend Snippet: DOCK11 and AGAP2 regulate ARF1 activity to promote the retrograde trafficking of HBV. ( A and B ) Co-immunoprecipitation of DOCK11 and AGAP2 with ARF1 in HepG2-NTCP-C4 cells (Endogenous) ( A ) or HepG2-NTCP-C4-Halo-DOCK11 cells that were transfected with the Halo-AGAP2 plasmid and treated with Dox (Exogenous) ( B ). ( C ) A deletion mutant of DOCK11, DHR2-DOCK11-Flag, was constructed and confirmed using immunoblotting analysis. ( D and E ) HepG2-NTCP-C4 cells were transfected with DHR2-DOCK11-Flag or full-length Halo-DOCK11 and then infected with NL-HBV particles. HBV DNA ( D ) and cccDNA ( E ) levels were detected by RTD-PCR. ( F and G ) Measurement of GEF activity. GEF activity of titrated recombinant DOCK11 protein toward a fixed protein concentration of recombinant CDC42 ( F ) or ARF1 ( G ) using the GTPase-Glo Assay. ( H ) GAP activity of titrated recombinant AGAP2 protein toward a fixed protein concentration of recombinant ARF1 using the GTPase-Glo Assay. ( I and J ) RAB7KO or control cells were infected with NL-HBV particles with or without BFA treatment for 30 hours. HBV DNA ( I ) and cccDNA ( J ) levels were detected by RTD-PCR. ( K ) The Golgi fraction was isolated from both groups of cells, and HBV capsid antigen was measured by enzyme-linked immunosorbent assay. ( L and M ) HepG2-NTCP-C4 cells were infected with NL-HBV particles for 30 hours together with NAV-2729, an inhibitor of ARF1, at the indicated concentration. HBV DNA ( L ) and cccDNA ( M ) levels were measured by RTD-PCR. Representative results from 3 independent experiments are shown. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. ( N ) Schematic representation of DOCK11-AGAP2-ARF1–mediated HBV retrograde trafficking via the EE-TGN-ER pathway to the nucleus.

Article Snippet: Arf1 Activation Assay Kit , Cell Biolabs, Inc , STA-407-1.

Techniques: Activity Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Mutagenesis, Construct, Western Blot, Infection, Recombinant, Protein Concentration, Glo Assay, Isolation, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hepatitis B Virus Utilizes a Retrograde Trafficking Route via the Trans-Golgi Network to Avoid Lysosomal Degradation

doi: 10.1016/j.jcmgh.2022.10.008

Figure Lengend Snippet: Key Resources

Article Snippet: Arf1 Activation Assay Kit , Cell Biolabs, Inc , STA-407-1.

Techniques: Subcloning, Purification, Recombinant, Protease Inhibitor, In Situ, Fluorescence, Activation Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Isolation, shRNA, Plasmid Preparation, Software