PMC

Confocal micrographs of HeLa cells double labeled with the anti–NDH II antibody and anti-Sp100 antibody (X103). (A) control cells; only some PML NBs colocalize with NDH II. (A1 and A2) Magnified views of indicated PML NBs show no colocalization (A1) or complete colocalization with NDH II (A2). The graph below shows the overlap of fluorescence intensity peaks along profiles spanning the PML NBs as indicated in the merged micrograph. (B) IFN-α–treated cells (1,000 U/ml for 24 h; +IFN-α); NDH II is recruited to PML NBs. (B1) A magnified view of indicated PML NBs shows complete colocalization with NDH II foci. The graph below shows the overlap of fluorescence intensity peaks along profiles spanning the PML NBs as indicated in the merged micrograph. (C and D) After inhibition of POL II transcription with α-amanitin (100 μg/ml for 3 h); (C) cells not stimulated with IFN-α (−IFN-α/+α-ama); (D) IFN-α–treated cells (1,000 U/ml for 24 h; +IFN-α/+α-ama). NDH II dissociates from PML NBs after inhibition of POL II transcription and is translocated to nucleolar periphery. (C1 and D1) Magnified views of indicated PML NBs show no colocalization with NDH II. (E and F) After treatment with AMD at concentration inhibiting only POL I transcription (0.02 μg/ml for 3 h); (E) cells not stimulated with IFN-α (−IFN-α/+AMD 0.02 μg/ml); (F) IFN-α–treated cells (1,000 U/ml for 24 h; +IFN-α/+AMD 0.02 μg/ml). Inhibition of POL I transcription does not cause NDH II dissociation from PML NBs and its translocation to nucleolar periphery. (E1 and F1) Magnified views of indicated PML NBs show no colocalization with NDH II in IFN-α–nonstimulated cells (E1), but complete colocalization in IFN-α–stimulated cells (F1). The graph below shows the overlap of fluorescence intensity peaks along profiles spanning the PML NBs as indicated in the merged micrograph. Bar, 2 μm.

Quantification of the colocalization of PML NBs and NDH II in control cells, in IFN-α–stimulated cells, and after inhibition of POL I transcription (0.02 μg/ml AMD; 3 h) performed in confocal images described in ( n = 20). Because there is no colocalization after inhibition of POL II transcription (100 μg/ml α-amanitin for 3 h; ) the quantification was not performed in this case. In control cells, NDH II colocalizes completely with 19% of PML NBs, 28% of PML NBs colocalize partially, and ∼53% do not colocalize with NDH II foci. In IFN-α–treated cells, NDH II colocalizes with 79% of PML NBs, ∼2% colocalize partially, and 19% of PML NBs do not colocalize with NDH II. After inhibition of POL I transcription, NDH II remains in the association with PML NBs and colocalizes with >75% of nuclear dots. Less than 5% of PML NBs colocalize partially, and 20% do not colocalize with NDH II.

Significant number of PML NBs associate with transcription sites after IFN-α induction. Quantification of the colocalization of PML NBs with NDH II and transcription sites in control and IFN-α–stimulated cells performed in confocal images described in (n = 20). In control cells, 75% of PML NBs do not contain either NDH II or nascent transcripts, ∼9% of bodies contain both NDH II and transcription sites, and 16% of bodies contain only NDH II; no bodies contain only transcription sites. After IFN-α treatment, the number of PML NBs colocalizing with transcription sites and NDH II increases up to 51%, whereas the number of PML NBs not containing either NDH II or nascent transcripts decreases to 30%.

NO slows down the H₂O₂killing of the katEG ndh nuo quadruple mutant.A, H₂O₂-alone and H₂O₂+NO sensitivity of the quadruple katEG ndh nuo mutant. B, a representative gel illustrating chromosome fragmentation patterns caused by H₂O₂-alone and H₂O₂+NO treatments in the katEG ndh nuo quadruple mutant. C, quantification of the chromosomal DNA loss in the katEG ndh nuo quadruple mutant in response to H₂O₂-alone or H₂O₂+NO treatments, from several gels like in "B". D, the effect of iron chelation with DF on H₂O₂-alone or H₂O₂+NO sensitivity of the katEG ndh nuo mutant. E, a representative gel illustrating the effect of iron chelation with DF on the chromosome fragmentation in the katEG ndh nuo mutant treated with either H₂O₂-alone or with H₂O₂+NO for 45 min. F, quantification of the chromosomal DNA loss from several gels like in "E". G, comparison of H₂O₂-alone killing patterns of the WT, katEG double, ndh nuo double, and the katEG ndh nuo quadruple mutants. H, comparison of H₂O₂+NO killing patterns of the WT, katEG double, ndh nuo double, and the katEG ndh nuo quadruple mutants. I, qualitative parameters of 2.5 mM H₂O₂ toxicity of H₂O₂-alone versus H₂O₂+NO treatments in various mutants. I, mode-one killing; II, mode-two killing. DF, deferoxamine; H₂O₂/HP, hydrogen peroxide; NO, nitric oxide.