Figure 3.
IFN-independent, IRF3-dependent regulation of ISG15 recapitulated in IRF3 knockout (KO) CRISPR/Cas-9 engineered telomerase immortalized (hTERT) HFs. (a) IRF3 KO clones generated by CRISPR/Cas-9 were screened by immunoblot for IRF3 alongside the parental Cas-9 hTERT HFs with GAPDH as a loading control. The dashed boxes denote which clones were chosen for further experiments. (b) Supernatants were collected from the Cas-9 hTERT HFs and individual clones after 24 h of HCMV infection (MOI 3) before IFNβ was quantified by an enzyme-linked immunosorbent assay (ELISA) (N.D: not detected indicates IFNβ levels less than the lower limit of quantification 5 pg/mL). (c) Successful IRF3 KO clones 7, 17 and 20 were infected with HCMV (MOI of 3) or treated with recombinant IFNβ (100U) in parallel with unsuccessful clone 6, and the parental Cas-9 hTERT HFs. Protein lysates were extracted at 24 h.p.i. before immunoblotting for ISG15 and GAPDH. (d) Individual clones and parental Cas-9 hTERT HFs were treated with supernatant from HCMV infected cells for 24 h before immunoblotting for ISG15 and GAPDH.