Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 8. Recruitment of DFCP1 into the proximity of mitochondria for HCV NS5A-induced mi- tophagy: (A) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-DFCP1, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-DFCP1 cells. Then, Huh7/RFP- LC3/Mito-miRFP670/GFP-DFCP1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A- mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. (B) The number of GFP-DFCP1 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. (C) The frames of selected live images show the magnified area in the white dashed box of the top panel in (A). The white arrowheads indicate the recruitment of GFP-DFCP1 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. (D) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-DFCP1, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-DFCP1 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-DFCP1 cells were transduced with (+) or without (−) pTRIP-

Article Snippet: Moloney murine leukemia virus (MMLV)-based retrovirus expression plasmids, including pMXs-IP-EGFP-mAtg5 (#38196), pMXs-puro GFP-DFCP1 (#38269), pMXs-IP GFP-Atg14 (#38264), and pMXs-IP-EGFP-ULK1 (#38193) were obtained from Addgene (Watertown, MA, USA).

Techniques: Transduction, Expressing, Confocal Microscopy, Labeling

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 9. Translocation of ATG14 into close proximity to mitochondria for HCV NS5A-induced mitophagy: (A) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG14, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-ATG14 cells. Then, Huh7/RFP- LC3/Mito-miRFP670/GFP-ATG14 cells were transduced with (+) or without (−) pTRIP-HCV NS5A- mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. (B) The number of GFP-ATG14 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequester Mito- miRFP670-expressing mitochondria, was quantified. (C) The selected live imaging frames show the magnified area in the white dashed box of the top panel in (A). The white arrowheads indicate the recruitment of GFP-ATG14 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. (D) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG14, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG14 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG14 cells were transduced with (+) or without (−)

Article Snippet: Moloney murine leukemia virus (MMLV)-based retrovirus expression plasmids, including pMXs-IP-EGFP-mAtg5 (#38196), pMXs-puro GFP-DFCP1 (#38269), pMXs-IP GFP-Atg14 (#38264), and pMXs-IP-EGFP-ULK1 (#38193) were obtained from Addgene (Watertown, MA, USA).

Techniques: Translocation Assay, Transduction, Expressing, Confocal Microscopy, Imaging, Labeling

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 10. Translocation of ULK1 into the proximity of mitochondria for HCV NS5A-activated mitophagy: (A) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses express- ing GFP-ULK1, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-ULK1 cells. Then, Huh7/RFP- LC3/Mito-miRFP670/GFP-ULK1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A- mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. (B) The number of GFP-ULK1 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. (C) The selected live imaging frames show the magnified area in the white dashed box of the top panel in (A). The white arrowheads indicate the recruitment of GFP-ULK1 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. (D) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ULK1, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-ULK1 cells. Then, Huh7/RFP- Parkin/Mito-miRFP670/GFP-ULK1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. (E) The number of GFP-ULK1 molecules recruited to the RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. (F) The selected live imaging frames show the magnified area in the white dashed box of the top panel in (D). The white arrowheads indicate the recruitment of GFP-ULK1 to Mito-miRFP670-labeled mitochondria after RFP-Parkin translocation. The data shown in (B,E) represent the mean ± SEM (n = 10, *** p < 0.001).

Article Snippet: Moloney murine leukemia virus (MMLV)-based retrovirus expression plasmids, including pMXs-IP-EGFP-mAtg5 (#38196), pMXs-puro GFP-DFCP1 (#38269), pMXs-IP GFP-Atg14 (#38264), and pMXs-IP-EGFP-ULK1 (#38193) were obtained from Addgene (Watertown, MA, USA).

Techniques: Translocation Assay, Transduction, Confocal Microscopy, Expressing, Imaging, Labeling

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Recruitment of ATG5 into close proximity to mitochondria for HCV NS5A-induced mitophagy: ( A ) Huh7 cells were transduced with lentiviruses expressing RFP-LC3 and mito-miRFP670 to establish Huh7/RFP-LC3/Mito-miRFP670 cells. Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG5, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-ATG5 cells. Then, Huh7/RFP-LC3/Mito-miRFP670/GFP-ATG5 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( B ) The number of GFP-ATG5 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. ( C ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the recruitment of GFP-ATG5 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. ( D ) Huh7 cells were transduced with lentiviruses expressing RFP-Parkin and Mito-miRFP670 to establish Huh7/RFP-Parkin/Mito-miRFP670 cells. Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG5, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG5 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG5 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( E ) The number of GFP-ATG5 molecules recruited to RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. ( F ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( D ). The white arrowheads indicate the recruitment of GFP-ATG5 to Mito-miRFP670-labeled mitochondria after translocation by RFP-Parkin. The data shown in ( B , E ) represent the mean ± SEM ( n = 10, *** p < 0.001).

Article Snippet: Moloney murine leukemia virus (MMLV)-based retrovirus expression plasmids, including pMXs-IP-EGFP-mAtg5 (#38196), pMXs-puro GFP-DFCP1 (#38269), pMXs-IP GFP-Atg14 (#38264), and pMXs-IP-EGFP-ULK1 (#38193) were obtained from Addgene (Watertown, MA, USA).

Techniques: Transduction, Expressing, Confocal Microscopy, Imaging, Labeling, Translocation Assay

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Recruitment of DFCP1 into the proximity of mitochondria for HCV NS5A-induced mitophagy: ( A ) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-DFCP1, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-DFCP1 cells. Then, Huh7/RFP-LC3/Mito-miRFP670/GFP-DFCP1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( B ) The number of GFP-DFCP1 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. ( C ) The frames of selected live images show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the recruitment of GFP-DFCP1 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. ( D ) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-DFCP1, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-DFCP1 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-DFCP1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( E ) The number of GFP-DFCP1 molecules recruited to RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. ( F ) The frames of selected live images show the magnified area in the white dashed box of the top panel in ( D ). The white arrowheads indicate the recruitment of GFP-DFCP1 to Mito-miRFP670-labeled mitochondria after translocation by RFP-Parkin. The data shown in ( B , E ) represent the mean ± SEM ( n = 10, *** p < 0.001).

Article Snippet: Moloney murine leukemia virus (MMLV)-based retrovirus expression plasmids, including pMXs-IP-EGFP-mAtg5 (#38196), pMXs-puro GFP-DFCP1 (#38269), pMXs-IP GFP-Atg14 (#38264), and pMXs-IP-EGFP-ULK1 (#38193) were obtained from Addgene (Watertown, MA, USA).

Techniques: Transduction, Expressing, Confocal Microscopy, Labeling, Translocation Assay

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Translocation of ATG14 into close proximity to mitochondria for HCV NS5A-induced mitophagy: ( A ) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG14, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-ATG14 cells. Then, Huh7/RFP-LC3/Mito-miRFP670/GFP-ATG14 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( B ) The number of GFP-ATG14 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequester Mito-miRFP670-expressing mitochondria, was quantified. ( C ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the recruitment of GFP-ATG14 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. ( D ) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG14, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG14 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG14 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( E ) The number of GFP-ATG14 molecules recruited to RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. ( F ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( D ). The white arrowheads indicate the recruitment of GFP-ATG14 to Mito-miRFP670-labeled mitochondria after RFP-Parkin translocation. The data shown in ( B , E ) represent the mean ± SEM ( n = 10, *** p < 0.001).

Article Snippet: Moloney murine leukemia virus (MMLV)-based retrovirus expression plasmids, including pMXs-IP-EGFP-mAtg5 (#38196), pMXs-puro GFP-DFCP1 (#38269), pMXs-IP GFP-Atg14 (#38264), and pMXs-IP-EGFP-ULK1 (#38193) were obtained from Addgene (Watertown, MA, USA).

Techniques: Translocation Assay, Transduction, Expressing, Confocal Microscopy, Imaging, Labeling

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Translocation of ULK1 into the proximity of mitochondria for HCV NS5A-activated mitophagy: ( A ) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ULK1, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-ULK1 cells. Then, Huh7/RFP-LC3/Mito-miRFP670/GFP-ULK1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( B ) The number of GFP-ULK1 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. ( C ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the recruitment of GFP-ULK1 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. ( D ) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ULK1, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-ULK1 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-ULK1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( E ) The number of GFP-ULK1 molecules recruited to the RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. ( F ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( D ). The white arrowheads indicate the recruitment of GFP-ULK1 to Mito-miRFP670-labeled mitochondria after RFP-Parkin translocation. The data shown in ( B , E ) represent the mean ± SEM ( n = 10, *** p < 0.001).

Article Snippet: Moloney murine leukemia virus (MMLV)-based retrovirus expression plasmids, including pMXs-IP-EGFP-mAtg5 (#38196), pMXs-puro GFP-DFCP1 (#38269), pMXs-IP GFP-Atg14 (#38264), and pMXs-IP-EGFP-ULK1 (#38193) were obtained from Addgene (Watertown, MA, USA).

Techniques: Translocation Assay, Transduction, Expressing, Confocal Microscopy, Imaging, Labeling