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Fig. 5
Hif-1α mediated anti-mycobacterial effect is iNOS dependent.
(A) Fluorescent confocal micrographs of DAF-FM DA stained embryos at 2 dpf in the absence of infection. Neutrophils are identified by lyz:dsRed expression. DAF-FM DA staining between the timepoint of infection and 1 dpi produced varying levels of background and stained cells of the central nervous system (neurons and notochord) as well as having leukocyte-associated staining. The line of staining at the top of each image is DAF-FM DA staining in the notochord. Punctae of DAF-FM DA show upregulation of NO signaling Dominant active hif-1αb (DA1) embryos had more punctae than phenol red (PR) controls. nos2a morpholino (NOS2MO) reduced the punctae in both the PR and DA1 background. (B) Fluorescent confocal micrographs of DAF-FM DA stained embryos at 2 dpf in the presence of Mm infection. In phenol red (PR) controls DAF-FM DA punctae are increased. DAF-FM DA staining is not specific for iNOS (it is a pan-NOS probe), and the nos2a morpholino (NOS2MO) was not able to downregulate all of the DAF-FM DA staining after Mm infection, although punctae number were reduced. The number of punctae was also reduced in the dominant active hif-1αb (DA1) injected embryos after Mm infection. Dominant negative hif-1αb (DN1) caused no change in punctae in the presence of Mm infection compared to PR controls. (C) Fluorescent confocal micrographs of iNOS antibody staining in mpx:GFP embryos. Phenol red (PR) injected controls had very low levels of anti-iNOS antibody staining. Dominant active hif-1αb (DA1) had increased levels of anti-iNOS antibody, a stain which was mainly neutrophil specific. (D) Bacterial burden at 4 dpi after injection of DA hif-1αb (DA1) or phenol red control (PR) and treatment with the pan-NOS inhibitor L-NAME. Data shown are mean ± SEM, n = 62–89 as accumulated from 3 independent experiments. (E) Bacterial burden at 4 dpi after injection of DA hif-1αb (DA1) and treatment with the iNOS inhibitor L-NIL. Data shown are mean ± SEM, n = 60–87 as accumulated from 3 independent experiments. (F) Bacterial burden at 4 dpi after co-injection of DA hif-1αb and the nos2a morpholino, using the standard control (SCMO) morpholino as a negative control. Data shown are mean ± SEM, n = 109–116 as accumulated from 4 independent experiments.