HIRA localizes at PML-NBs in response to exogenous IFN stimulation in a cell type and CDK-dependent manner.

Primary, immortalized, or carcinoma cells were mock treated or stimulated with IFN-β or IFN-γ (100 IU/ml) as indicated. Cell monolayers were fixed and permeabilized at 24 h post-stimulation (unless stated otherwise). (A, B) Nuclear localization of HIRA or Daxx (green, as indicated) with respect to PML (red) in mock, IFN-β, or IFN-γ treated (as indicated) HFt cells. Nuclei were stained with DAPI (blue). Cut mask (yellow) highlights regions of colocalization between HIRA or Daxx and PML (as indicated). Weighted colocalization coefficients shown. (C) Quantitation of the population of HFt cells positive for HIRA localization at PML-NBs in mock or IFN treated HFt cells (as shown in A). n ≥ 200 cells per sample population derived from four independent treatments. Means and SD shown. (D) Quantitation of the population of cells positive for HIRA localization at PML-NBs in mock or IFN-β treated HFt cells at 4, 8, or 24 h post-treatment. n ≥ 300 cells per sample population derived from four independent treatments. Means and SD shown. (E) Quantitation of HIRA or Daxx colocalization with PML in HFt cells (as shown in A, B). Boxes: 25th to 75th percentile range; black line: median weighted (w.) colocalization coefficient; whiskers: 5th to 95th percentile range; dashed line: coincident threshold (weighted colocalization coefficients HIRA or Mx1 mRNA levels in mock or IFN-β treated HFt cells at 8 or 24 h post-treatment. Values normalized to 8 h no IFN. n = 3, means and SD shown. (G) Quantitation of the population of cells positive for HIRA localization at PML-NBs in mock or IFN-β treated primary (IMR-90, MRC5, HFs), immortalized (MRC5t, HFt, RPE, HaCat), or carcinoma (U2OS, SAOS, HeLa, A549) cells. n ≥ 300 cells per sample population derived from four independent treatments. Means and SD shown. Representative images for each cell line -/+ IFN-β treatment are shown in S5 Fig. (H) qRT-PCR quantitation of HIRA mRNA levels in unstimulated primary, immortalized, or carcinoma cells (as in G). Values normalized to the levels of HIRA mRNA in primary IMR-90 cells. Mean (RQ) and SD (RQ min/max) shown. (I) Nuclear localization of HIRA (green) and PML (red) in HFt cells pre-treated with DMSO (carrier control), CHIR-99021 HCl (GSK-3α/β inhibitor; GSKi, 250 nM), or flavopiridol (CDK inhibitor; CDKi, 50 nM), for 1 h prior to IFN-β stimulation in the presence of drug for 24 h. Cut mask (yellow) highlights regions of colocalization between HIRA and PML. Weighted colocalization coefficients shown. (J, K) Quantitation of the population of cells positive for HIRA localization at PML-NBs following treatment with DMSO, GSKi, or CDKi over a range of concentrations (as indicated) prior to stimulation (or not) with IFN-β in the presence of drug. n≥ 400 cells per sample population derived from a minimum of four independent treatments. Means and SD shown. ** P P U-test.