Journal: Nature Communications
Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication
Figure Lengend Snippet: HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)
Article Snippet: Purified total RNA samples were tested for HCV RNA expression (Primer Design Ltd, UK) in a qRT-PCR as per the manufacturer’s guidelines (CellsDirect Kit; Invitrogen, UK) in an MxPro-3000 PCR machine (Aligent, US).
Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY