Fig 1: Axl kinase augments ZIKV infection of HTO. (A) Morphology and viability evaluation of HTO. Bright-field imaging (top) and LIVE/DEAD Confocal 3D Z-Stack projection (bottom) of representative organoids after post-thaw recovery in culture (red fluorescence depicts dead cells and green depicts live cells). Bars, 100 μm. (B and C) HTO were infected with ZIKV in the presence or absence of R428 (1 μM) and at 24 h and 72 h postinfection, ZIKV RNA copies (B) and progeny titers in the supernatant (C) were determined using qRT-PCR (30 HTO per data point) and plaque assay (10 HTO per data point), respectively. (D) Viability of R428-treated and untreated HTO was determined using CellTiter Glo 3D Cell Viability Assay at 72 h postinfection (values are averages ± SD [error bars] from ≥6 HTO per group). (E) Expression of IFNB1, IFIT1, and MxA transcripts was determined using qRT-PCR and expressed as mRNA fold change to mock-infected cells by normalizing to GAPDH (values are means ± SEM [error bars} of four data points, and each data point is a pool of RNA from 30 HTO). HTO were infected with 105 PFU, and DMSO vehicle was used as the untreated control for all assays. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 2: The antiviral state of LC is elevated compared to that of SC under basal conditions. (A) IFIT1 mRNA expression in mock-infected and ZIKV-infected SC and LC reported as a change in cycle threshold (ΔCt) between GAPDH and IFIT1 transcripts 24 h after ZIKV infection (MOI of 1). Values are means ± SD (error bars) for 4 to 6 independent experiments. (B) The effect of ZIKV infection on IFIT1 protein expression was evaluated in SC and LC at 48 h postinfection (MOI of 1) by staining for ZIKV (anti-ZIKV-E [green]) and IFIT1 (anti-IFIT1 [red]). (C) IFIT1 protein localization following infection was evaluated by merging nucleus staining (DAPI [blue]) with IFIT1 staining (anti-IFIT1 [red]). Nuclear localization of IFIT1 is indicated by overlapping blue and red staining (magenta). (D) mRNA expression of antiviral genes—type I IFNs (IFNA2 and IFNB1), type III IFNs (IFNL1 and IFNL3), and IFIT1 and MxA—was determined and compared between naive SC and LC using qRT-PCR and reported as ΔCt between GAPDH and target gene (tg) transcripts. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Fig 3: Axl suppresses the antiviral state of human SC. (A) Protein expression of total and phosphorylated STAT1 (p-STAT1), SOCS1, and SOCS3 was determined in total cell lysates from mock-infected and ZIKV-infected (MOI of 10) SC following R428 treatment (1 μM) by Western blotting. β-Actin was used as a housekeeping loading control. Each lane shows the results from an independent experiment. SC with or without R428 treatment (1 μM) were infected with ZIKV at an MOI of 1 and at 30 h postinfection. (B) ZIKV RNA copies were determined by qRT-PCR and expressed as ZIKV PFU equivalents per microgram of RNA. (C) mRNA fold change of ISGs (IFIT1, MxA, IFI6, ISG15, ISG20, IFITM3, IFIH1, and DHX58), type I IFN genes (IFNA2, IFNA5, and IFNB1), and type III IFN genes (IFNL1 and IFNL3) compared to mock-infected cells were measured using qRT-PCR by normalizing to GAPDH. (D) ZIKV RNA copies were also determined in SC following R428 treatment (1 μM) and infection with an MOI of 1 and an MOI of 10 at 24 h. (E) The effect of infectious dose on the mRNA expression of IFIT1, MxA, IFNL1, and IFNB1 was determined in SC with and without R428 treatment by normalizing to GAPDH (values are means ± standard errors of the means [SEM] [error bars] from three independent experiments). (F) Secreted IFN-β protein levels in SC supernatant at 24 h postinfection was determined by ELISA. The dotted line indicates the minimal detection limit of the assay. (G and H) The effect of R428 treatment (1 μM) on total and phosphorylated IRF3 (p-IRF3) protein expression (G) and total STAT2 protein expression (H) was determined in both mock-infected and infected SC at 24 h postinfection (MOI of 10) by Western blotting. DMSO vehicle was used as untreated control for all assays. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Supplier Page from Abcam for Anti-IFIT1 antibody