Fig. 7

Dominant negative Hif-2α decreases bacterial burden via an iNOS dependent mechanism.

(A) Example fluorescence confocal z-stacks of the caudal vein region of embryos stained with anti-nitrotyrosine antibody, imaged at 1 dpi (2 dpf), in the absence and presence of Mm infection. Embryos were injected with dominant negative hif-2αa (DN2), dominant active hif-2&aklpha;a (DA2), or phenol red (PR). (B) Corrected fluorescence intensity levels of anti-nitrotyrosine antibody confocal z-stacks in uninfected larvae. Dominant negative hif-2αa (DN2) had significantly increased anti-nitrotyrosine levels in the absence of Mm bacterial challenge compared to phenol red (PR) injected controls. Data shown are mean ± SEM, n = 42–92 cells accumulated from 5 embryos. Graph shown is a representative dataset of 3 independent experiments. (C) Corrected fluorescence intensity levels of anti-nitrotyrosine antibody confocal z-stacks of dominant active hif-2αa (DA2), or phenol red (PR) control injected embryos in the presence or absence of Mm infection at 1 dpi (2 dpf). Data shown are mean ± SEM, n = 46–92 cells accumulated from 5 embryos. Graph shown is a representative dataset of 3 independent experiments. (D) Bacterial burden at 4 dpi after injection of dominant negative hif-2αa (DN2) or phenol red control (PR) and treatment with the pan-NOS inhibitor L-NAME. Data shown are mean ± SEM, n = 67–85 as accumulated from 3 independent experiments. (E) Bacterial burden at 4 dpi after injection of dominant negative hif-2αa (DN2) and treatment with the iNOS inhibitor L-NIL. Data shown are mean ± SEM, n = 52–58 as accumulated from 3 independent experiments. (F) Bacterial burden at 4 dpi after co-injection of dominant negative hif-2αa (DN2) and the nos2a morpholino, using the standard control (SC) morpholino as a negative control. Data shown are mean ± SEM, n = 103–109 as accumulated from 4 independent experiments. (G) Schematic of the effect of early Hif-α modulation during early Mm infection.