Journal: PLoS ONE

Article Title: Reticulon-3 modulates the incorporation of replication competent hepatitis C virus molecules for release inside infectious exosomes

doi: 10.1371/journal.pone.0239153

Figure Lengend Snippet: ( A&B ) Reticulon 3L&S (RTN3L&S) was knockdown using specific siRNA( A ) and LentiCRISPR lentiviral CRISPR/Cas9 ( B ) methods in HCV FL-Replicon cells alongside appropriate control as indicated. 48h post RTN3 knockdown, cell culture supernatants, and cells were harvested. Total proteins extracted from cells were subjected to western blotting probing for RTN3L&S, NS5A, and NS3 with β-actin serving as an equal loading control. ( C ) NanoSight NS300 was used to quantify cell released exosomes in cell culture supernatants. ( D ) Cell released exosomes from LentiCRISPR lentiviral CRISPR/Cas9 knockdown of RTN3L&S conditioning and was co-cultured with naïve Huh7.0 cells (5k Exosomes: 1 Huh7.0 cell) over 72h. Total RNA was then extracted from cells and analyzed by RT-qPCR for HCV RNA using 18s as a housekeeping gene. Data is representative of 3 independently repeat experiments with */ # p<0.05 was considered significant by Mann–Whitney U test.

Article Snippet: The complementary oligonucleotides for guide RNAs (gRNAs) were annealed and cloned into the LentiCrispr V2 vector [LentiCrispr, a gift from Feng Zhang; Addgene plasmid # 52961; http://n2t.net/addgene:52961 ; RRID: Addgene_52961)] [ ].

Techniques: Knockdown, CRISPR, Control, Cell Culture, Western Blot, Quantitative RT-PCR, MANN-WHITNEY