Journal: JHEP Reports

Article Title: miR-22 inhibition reduces hepatic steatosis via FGF21 and FGFR1 induction

doi: 10.1016/j.jhepr.2020.100093

Figure Lengend Snippet: OCA simultaneously induces miR-22 , as well as FGF21, FGFR1, and PGC1α in liver cells. Inhibiting miR-22 enhances OCA-induced FGF21 signaling leading to AMPK and ERK1/2 activation. miR-22, FGF21, FGFR1, and PGC1α mRNA (A) and protein (B) levels in Huh7 cells treated with DMSO or OCA for 6 h. (C) Protein levels in Huh7 cells infected with adenovirus negative control or the miR-22 inhibitor followed by DMSO or OCA (5 μM) treatment for 6 h. (D) miR-22 as well as the indicated mRNA levels in PHHs treated with DMSO or OCA for 6 h. (E) Indicated mRNA levels in PHHs infected with adenovirus negative control or the miR-22 inhibitor followed by DMSO or OCA (5 μM) treatment for 6 h. RT-PCR was performed in triplicate for each sample. PHHs were isolated from 1 donor liver. Data are presented as the mean ± SD (n = 3). One-way ANOVA with Tukey’s t test. ∗ p <0.05, ∗ ∗p <0.01, ∗∗ ∗p <0.001. OCA, obeticholic acid; PHHs, primary human hepatocytes; RT-PCR, reverse transcription PCR.

Article Snippet: Primary human hepatocytes (PHHs) (Sekisui XenoTech LLC; Cat. CHP06, Kansas City, KS, USA) were maintained in OptiCulture hepatocyte media (Sekisui XenoTech LLC).

Techniques: Activation Assay, Infection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Isolation, Reverse Transcription

Journal: The American Journal of Pathology

Article Title: Chaperone-Mediated Autophagy Promotes Beclin1 Degradation in Persistently Infected Hepatitis C Virus Cell Culture

doi: 10.1016/j.ajpath.2018.06.022

Figure Lengend Snippet: Hepatitis C virus (HCV) infection activates endoplasmic reticulum (ER) stress response and induces expression of unfolded protein response (UPR) genes in infected primary human hepatocytes (PHHs) and Huh-7.5 cells. A: Cell lysates prepared from infected PHHs were examined for the expression of ER-stress chaperone, binding immunoglobulin protein (BiP), and expression levels of protein kinase RNA-activated–like endoplasmic reticulum kinase (PERK), activating transcription factor-6α (ATF6α), and inositol-requiring enzyme-1α (IRE1α) by Western blot analysis. B: Cell lysates of HCV-infected Huh-7.5 cells were prepared at different days, and the expression levels of PERK, IRE1α, and ATF6α were examined by Western blot analysis. C: mRNA levels of PERK in infected Huh-7.5 cells at different days measured by real-time quantitative RT-PCR (RT-qPCR). D: mRNA levels of IRE1α in infected Huh-7.5 cells at different days measured by real-time RT-qPCR. E: mRNA levels of ATF6α in infected Huh-7.5 cells at different days measured by real-time RT-qPCR. ∗∗P < 0.01, ∗∗∗P < 0.001 versus day 0.

Article Snippet: Cell Culture, Antibodies, and Chemicals Nontransformed primary human hepatocytes (PHHs) were supplied by XenoTech LLC, Kansas City, MO.

Techniques: Virus, Infection, Expressing, Binding Assay, Western Blot, Quantitative RT-PCR

Journal: The American Journal of Pathology

Article Title: Chaperone-Mediated Autophagy Promotes Beclin1 Degradation in Persistently Infected Hepatitis C Virus Cell Culture

doi: 10.1016/j.ajpath.2018.06.022

Figure Lengend Snippet: Persistent hepatitis C virus (HCV) infection activates nuclear factor erythroid 2–related factor (Nrf2) that induces chaperone-mediated autophagy (CMA). A: The human lysosome-associated membrane protein 2A (LAMP2A) and heat shock cognate 70 kDa (Hsc70) promoter sequence were examined for the presence of antioxidant response element (ARE) consensus sequences (TGAnnnnGC) indicated by arrowheads and ARE-like sequences (TGAnnnGC or TGAnnnnnGC) shown by arrows. B: Western blot analysis shows expression levels of Nrf2, phosphorylated Nrf2 (pNrf2), Hsc70, and LAMP2A in infected primary human hepatocytes (PHHs) during 15 days. C: Expression of Hsc70 levels in HCV-infected Huh-7.5 cells by immunostaining. D: Expression of LAMP2A in HCV-infected Huh-7.5 cells by immunostaining of cytospin slides at different days. E: Quantification of cells expressing Hsc70 by ImageJ version 1.52c. F: Quantification of LAMP2A-positive cells by ImageJ. G:Hsc70 and LAMP2A mRNA levels in HCV-infected Huh-7.5 cells measured by real-time quantitative RT-PCR. H: Western blot shows expression of LAMP2A, Hsc70, and β-actin after siRNA-mediated silencing of Nrf2 in HCV-infected Huh-7.5 cells. I: MTT assay showing viability of HCV-infected Huh-7.5 cells with or without silencing LAMP2A. J: MTT assay showing viability of HCV-infected Huh-7.5 cells with or without Nrf2 silencing. ∗P < 0.05, ∗∗∗P < 0.001 versus day 0. Scale bars = 200 μm (C and D).

Article Snippet: Cell Culture, Antibodies, and Chemicals Nontransformed primary human hepatocytes (PHHs) were supplied by XenoTech LLC, Kansas City, MO.

Techniques: Virus, Infection, Membrane, Sequencing, Western Blot, Expressing, Immunostaining, Quantitative RT-PCR, MTT Assay

Journal: The American Journal of Pathology

Article Title: Chaperone-Mediated Autophagy Promotes Beclin1 Degradation in Persistently Infected Hepatitis C Virus Cell Culture

doi: 10.1016/j.ajpath.2018.06.022

Figure Lengend Snippet: Chaperone-mediated autophagy (CMA) targets beclin1 degradation in hepatitis C virus (HCV) infection. A: Schematic representation showing the presence of multiple pentapeptide motif (KFERQ-like), CMA motifs, and their location in beclin1 protein sequences. B: Western blot shows beclin1 and lysosome-associated membrane protein 2A (LAMP2A) expression in Huh-7.5 cells after serum starvation. C: Beclin1 expression in HCV-infected primary human hepatocytes (PHHs). D: Beclin1 expression in HCV-infected Huh-7.5 cells. E: Beclin1 mRNA levels in HCV-infected Huh-7.5 cells by real-time quantitative RT-PCR. F: Western blot analysis shows silencing LAMP2A expression restores beclin1 expression without altering HCV replication in infected Huh-7.5 cells. G: Co-immunoprecipitation shows interaction among beclin1, LAMP2A, and heat shock cognate 70 kDa (Hsc70) in uninfected and HCV-infected Huh-7.5 cells at 6 days and 21 days. H: Sulforaphane induces nuclear factor erythroid 2–related factor (Nrf2) activation and its nuclear translocation in uninfected Huh-7.5 cells. I: Western blot analysis demonstrating expression of LAMP2A and beclin1 in uninfected Huh-7.5 cells after treatment with increasing concentrations of sulforaphane. J: CMA functional assay showing lysosomal degradation of recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence and absence of protease inhibitors. K: Quantification of GAPDH bands in Western blot showing amount of GAPDH uptake and degradation during serum starvation and late HCV infection. ∗∗∗P < 0.001 versus day 0. Scale bars = 200 μm.

Article Snippet: Cell Culture, Antibodies, and Chemicals Nontransformed primary human hepatocytes (PHHs) were supplied by XenoTech LLC, Kansas City, MO.

Techniques: Virus, Infection, Western Blot, Membrane, Expressing, Quantitative RT-PCR, Immunoprecipitation, Activation Assay, Translocation Assay, Functional Assay, Recombinant

Journal: Protein & Cell

Article Title: Identification of serotonin 2A receptor as a novel HCV entry factor by a chemical biology strategy

doi: 10.1007/s13238-018-0521-z

Figure Lengend Snippet: 5-HT 2A R plays a role in HCV infection. (A) The topology of 5-HT 2A R. (B) Expression of 5-HT 2A R, HCV core and GADPH in HCV-infected or mock Huh7.5.1 cells. HCVcc at 2 MOI was used to infected cells over the course of 10 days. The expression of host GADPH is shown as a control protein. (C) Silencing of 5-HT 2A R and CD81 impairs HCV infection. Huh7.5.1 cells were silenced with sh-NC, sh-5HT 2A R-3 or sh-CD81, followed by HCVcc infection over the course of 5 days. The transcript levels of 5-HT 2A R and CD81 were quantified by qRT-PCR, normalized to GAPDH and graphed as a percentage of the maximum number of copies determined in sh-NC-containing cells. All results are graphed as the mean ± SD for triplicate samples. (D) The infection of HCVcc, but not VSV-Gpp, is correlated with 5-HT 2A R expression. Huh7.5.1 cells containing shRNAs or sh-NC were infected with HCVcc or VSV-Gpp at 37 °C for 48 h. The transcript levels were quantified by qRT-PCR, normalized to GAPDH and graphed as a percentage of copies determined in sh-NC-containing cells. The infections were quantified by measuring the luciferase activity in relative luminescence units. Virus infection is expressed as a percentage relative to that in sh-NC-containing cells. (E) HCVcc infection is rescued by 5-HT 2A R overexpression. Huh7.5.1 cells containing sh-NC, sh-5HT 2A R and pcDNA empty plasmid, sh-5HT 2A R and p5HT 2A R wt , or sh-5HT 2A R and p5HT 2A R shRes were infected by HCVcc at 37 °C for 48 h. The expression levels of 5-HT 2A R were examined by Western blot and normalized to GADPH. Virus infection and protein expression are expressed as a percentage relative to sh-NC-containing cells. (F) Huh7.5.1 cells infected by HCVcc in the presence of antibodies or blocking peptide at 50 μg/mL at 37 °C for 48 h. Virus infection is expressed as percentages relative to buffer-treated control cells. (G) PBZ inhibits HCVcc in Huh7.5.1, Huh7 and PHHs. All cells infected by HCVcc were treated with PBZ at the indicated concentrations at 37 °C for 48 h. HCV RNAs are quantified by qRT-PCR and expressed as percentages relative to 0.5% DMSO-treated control cells. (H) Silencing of 5-HT 2A R in Huh7.5.1, Huh7 and PHHs impair HCV infection. Huh7.5.1 cells, Huh7 cells and PHHs were silenced with sh-NC, sh-CD81 or sh-5HT 2A R, followed by HCVcc infection at 37 °C for 48 h. HCV RNAs are quantified by qRT-PCR and expressed as percentages relative to sh-NC-containing control cells. (I) Intracellular HCV genome levels detected in Huh7.5.1 cells, which are infected by virus containing the structural region of the indicated genotypes, treated with 10 μmol/L PBZ. The infections are quantified by measuring HCV RNAs for detection by qRT-PCR and expressed as percentages relative to 0.5% DMSO-treated cells. (J) 5-HT 2A R is required for HCV cell-to-cell spread. Cells containing sh-NC, sh-5HT 2A R and sh-CD81 were infected with HCV (JFH-1) with an EGFP reporter. At 24 hpi, the cells were washed and incubated in fresh medium containing 1% methyl cellulose. To examine the effect of anti-CD81 mAb to HCV cell-to-cell spread, Huh7.5.1 cells were infected by HCV (JFH-1) with an EGFP reporter. At 24 hpi, the cells were washed and incubated in fresh medium containing anti-IgG or anti-CD81 mAb together with 1% methyl cellulose. At 72 hpi, the number of EGFP-positive cells per foci was counted, and the size of the foci observed is expressed as the average percentage of total foci. All results are graphed as the mean ± SD for triplicate samples. The data presented are representative of three independent experiments

Article Snippet: Primary human hepatocytes (PHHs) were purchased from Wuhan Procell Corporation (China).

Techniques: Infection, Expressing, Control, Quantitative RT-PCR, Luciferase, Activity Assay, Virus, Over Expression, Plasmid Preparation, Western Blot, Blocking Assay, Incubation