Figure 4
From: Resistance to HSV-1 infection in the epithelium resides with the novel innate sensor, IFI-16
Cytoplasmic p204 neutralization results in an elevation in HSV-1 in the cornea. (a) To assess siRNA knockdown of p204, WT mice were transfected with either nonspecific siRNA or to p204. Twenty-four hours after transfection, knockdown was evaluated by confocal microscopy. White bar, 100 μm. (b) WT mice were infected with HSV-1 (1,000 p.f.u. per cornea) and subsequently administered a subconjunctival injection of isotype or anti-mouse p204 polyclonal antibody (10 μg) to neutralize cytosolic protein. Forty-eight hour pi corneas were harvested and evaluated for viral titer by plaque assay. Bars represent two experiments of four corneas per group and are expressed as the mean log p.f.u. per cornea±s.e.m. (c, left panels) To confirm the ability of the p204 antibody (red) to access cellular cytoplasm, WT mice were infected with HSV-1 and administered a subconjunctival p204 injection (lower panel) or PBS (upper panel). The corneas were then fixed and stained with a DyLight549-conjugated anti-rabbit secondary and DAPI (blue, nuclei). Images represent two independent experiments of 2–4 corneas per group and were imaged at a magnification of × 200. White bars, 100 μm. (panel c, right panels) To establish cellular location of the injected antibody, infected corneas were administered a subconjunctival injection of p204 antibody or PBS and were then stained with phalloidin (green, cell perimeters), p204 (red), and DAPI (blue) and imaged at × 600 with a digital zoom of 10. White bar, 10 μm; N, nuclei, yellow arrows, cytoplasmic location of p204 antibody. HSV, herpes simplex virus; PBS, phosphate-buffered saline; siRNA, small-interfering RNA; WT, wild type.
