Routine Western blot to check autophagic flux: Cautions and recommendations

Gómez-Sánchez, Rubén; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M. S.; Rodríguez-Arribas, Mario; Pedro, José Manuel Bravo-San; Fuentes, José; González‐Polo, Rosa A.

doi:10.1016/j.ab.2015.02.020

Cited by 25 publications

(19 citation statements)

References 18 publications

(1 reference statement)

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“…With the aim of verifying the lysosomal activity on VP2 proteins, we analyzed the fate of VP2 and VP3 in IBDV-infected QM7 cells employing bafilomycin A1 (BafA1), which specifically inhibits the proton pump (H ϩ -ATPase) responsible for intravesicular acidification and lysosomal protein degradation (32,33). First, we tested BafA1 treatment by assessing the lysosomal degradation of LC3-II in autophagosomes, which depends on lysosomal activity and has been previously reported to be disrupted by BafA1, causing an increase in the relative levels of both LC3-I precursor and LC3-II (34)(35)(36). Accordingly, we observed an increase in LC3-I and LC3-II after BafA1 treatment of QM7 cells (data not shown).…”

Section: Resultsmentioning

confidence: 67%

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication

Giménez

Zanetti

Terebiznik

et al. 2018

J Virol

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Birnaviruses are unconventional members of the double-stranded RNA (dsRNA) viruses group that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the viral encoded RNA-dependent RNA-polymerase (RdRp). This and other structural features suggests that birnaviruses may follow a completely different replication program from that followed by members of the family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here, we demonstrated that the (IBDV), a prototypical member of the family, hijacks endosomal membranes of infected cells through the interaction of viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extra-cellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, significantly diminished in VP3 P2 stably overexpressing cells. Altogether, our results indicate that the association of VP3 with endosomes has a relevant role in IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses. Infectious Bursal Disease (IBD, also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is the (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increases their susceptibility to other pathogens.IBDV is a member of family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3,with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by VP3 PATCH2 domain and demonstrated its relevant role in the context of viral infection.

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Section: Resultsmentioning

confidence: 67%

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication

Giménez

Zanetti

Terebiznik

et al. 2018

J Virol

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“…In brief, the increased expression of p62 in alveolar epithelial cells after H/R, especially Rap treatment may be explained by a high synthesis/ degradation ratio of p62. This also indicates it should be cautious to use p62 as an indicator of autophagy flux with caution and the level of p62 mRNA should be simultaneously measured along with the p62 protein level [41,44].…”

Section: Discussionmentioning

confidence: 99%

Autophagy Activation by Rapamycin Before Hypoxia-Reoxygenation Reduces Endoplasmic Reticulum Stress in Alveolar Epithelial Cells

Fan

Chen

Huang

et al. 2017

Cell Physiol Biochem

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Background: To determine potential effects of autophagy activation on hypoxia-reoxygenation (H/R) induced damage of a rat alveolar epithelial cell line. Methods: CCL149 cells were subjected to autophagy agonist (rapamycin, Rap), autophagy inhibitor (3-methyladenine, 3-MA) or PBS for 1 h before H/R treatment for 2 h, 4 h and 6 h. The optimal concentration of Rap (150 nM, 200 nM and 250 nM) or 3-MA (5 mM, 10 mM and 15 mM) was obtained from MTT assay. Autophagy was determined by fluorescence microscopy of eRFP-LC3 positive cells, transmission electron microscopy of autophagosome, western blot of LC3, AMPK, Beclin-1, HDAC6 and p62 proteins. Endoplasmatic reticulum stress was indicated by detecting expressions of BIP, XBP-1 and CHOP via western blot. Results: Rap at concentration of 250 nM before H/R increased the autophagy formation with more eRFP-LC3 positive cells and higher expressions of LC3-II, Beclin-1, HDAC6 and p62, but lower expressions of BIP, XBP-1 and CHOP in H/R treated CCL149. This effect seemed to be still obvious after H/R exposure for 6 h. The contrary results were obtained by treatment with 5 mM 3-MA. Conclusion: Rap might be a promising agent before mechanical ventilation or reperfusion to prevent re-damage in hypoxia related lung diseases.

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“…Bright field images were taken simultaneously and the number of fluorescing to total number of cells quantified. Cells were washed and collected on ice in 50 μL RIPA buffer, 32 containing proteinase inhibitors, for western blotting analysis. For comparison to Lipofectamine transfection ES-2 cells were cultured as above and incubated for 72 hrs in growth media containing 100 nM siRNA freshly prepared as either cESG-siRNA or Lipofectamine RNAiMAX-siRNA complexes (prepared according to the manufacturer’s directions).…”

Section: Methodsmentioning

confidence: 99%

Chemically Modified Dendritic Starch: A Novel Nanomaterial for siRNA Delivery

2015

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Nanostructured starches are naturally derived nanomaterials that can be chemically modified to allow for the introduction of functional groups, enhancing their potential for drug delivery and other biotechnology applications. In this proof of concept study, we investigate chemically modified, enzymatically synthesized glycogen (ESG) nanodendrites as a biodegradable, biocompatible, siRNA delivery system. Commercially available ESG was modified using glycidyltrimethylammonium chloride (GTMA), introducing quaternary ammonium groups via an epoxide ring opening reaction. This cationic ESG (cESG) electrostatically bound siRNA and successfully knocked-down protein expression in an in vitro ovarian clear cell carcinoma model. The construct exhibited sustained siRNA delivery for up to six days while exhibiting less toxicity than a common liposome-based siRNA delivery reagent, Lipofectamine RNAiMAX. These promising results set the stage for use of dendritic starch as a cost-effective, easily modifiable nanoscale delivery system for a diverse range of cargo including nucleic acids and therapeutic compounds.

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Routine Western blot to check autophagic flux: Cautions and recommendations

Cited by 25 publications

References 18 publications

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication

Autophagy Activation by Rapamycin Before Hypoxia-Reoxygenation Reduces Endoplasmic Reticulum Stress in Alveolar Epithelial Cells

Chemically Modified Dendritic Starch: A Novel Nanomaterial for siRNA Delivery

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