CRISPR-mediated KO of top scoring HDFs impairs CoV replication.
2021
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Zugriff:
(A) Immunofluorescence staining of MERS-CoV infected of Huh7 cells containing KO of top scoring HDFs. dsRNA is shown in green, and DAPI is shown in blue. (B) Western blot analysis quantification of LC3II and p62 expression. Protein expression is normalized to beta actin and relative to the untreated condition in WT and FKBP8-KO cell treated with rapamycin or infected with HCoV-229E. Quantification data can be found in Supporting information S1 Data , tab 15. Western blot raw images are depicted in S1 Raw Images. (C) Western blot analysis quantification of LC3II expression normalized to beta actin and relative to WT. Protein expression is depicted in TMEM41B-, MINAR1-, and FKBP8-KO cells after treatment with rapamycin and HCoV-229E infection. Statistical significance was determined using the Holm–Sidak method in GraphPad Prism. p -Values are indication, and p -values <0.05 are defined as statistically significant. Quantification data can be found in Supporting information S1 Data , tab 16. Western blot raw images are shown in S1 Raw Images. (D) Immunofluorescence staining of HCoV-229E, SARS-CoV, and SARS-CoV-2 infected Huh7 cells with TMEM41B, FKBP8, and MINAR1-KO, as well as a stable ACE2 expression. dsRNA is shown in green, DAPI is shown in blue, and ACE2 is shown in red. Scale bar is 50 μm. All images were acquired using an Evos Auto FL2 and processed in Fiji. (E) Relative cytotoxicity of TMEM41-KO, FKBP8-KO, and MINAR1-KO is depicted in %. Two-tailed unpaired Student t test was used to determine significance in GraphPad Prism 8.3.1. (F) Sanger sequencing of FKBP8-KO, MINAR1-KO, and TMEM41B-KO verifies Cas-9–mediated double-strand break in multiple alleles of the KO cells. PAM sequence is indicated in red, and binding site of sgRNA is indicated in blue. Used reagents are listed in detail in Table 1 . ACE2, angiotensin converting enzyme 2; CoV, coronavirus; FKBP8, FK506 binding protein 8; HCoV, human coronavirus; HDF, host dependency factor; KO, knockout; MERS-CoV, Middle East Respiratory Syndrome ...
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CRISPR-mediated KO of top scoring HDFs impairs CoV replication.
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Autor/in / Beteiligte Person: | Annika Kratzel (10492471) ; Jenna N. Kelly (10492462) ; Philip V’kovski (3716905) ; Jasmine Portmann (4053757) ; Yannick Brüggemann (11883854) ; Daniel Todt (777446) ; Nadine Ebert (10492480) ; Neeta Shrestha (11883857) ; Philippe Plattet (330823) ; Claudia A. Staab-Weijnitz (5326895) ; Albrecht von Brunn (29223) ; Eike Steinmann (32663) ; Ronald Dijkman (330307) ; Gert Zimmer (223023) ; Stephanie Pfaender (2868038) ; Volker Thiel (78076) |
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Veröffentlichung: | 2021 |
Medientyp: | Bild |
DOI: | 10.1371/journal.pbio.3001490.s010 |
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