nontransformed primary human hepatocytes (phhs)  (Sekisui XenoTech)

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