Intracellular Pathogen Leishmania donovani Activates Hypoxia Inducible Factor-1 by Dual Mechanism for Survival Advantage within Macrophage
Figure 2
LD promotes HIF-1α nuclear localization in macrophages in vitro and in vivo.
A. J774 cells were infected with LD (MOI-1∶10) (lower panel) or remain uninfected (upper panel). After 6 h of infection indirect immunofluorescence assay was performed using HIF-1α antibody. DAPI was used for nuclei staining of the host and intracellular LD (indicated by white arrows). In lower panel bigger and horizontal white arrow represents the J774 cell that was not infected with LD. Result is representative of one of six independent experiments. B. Peritoneal macrophages were isolated from BALB/c mice and infected with LD (MOI-1∶10). After 6 h of infection indirect immunofluorescence assay was performed using HIF-1α antibody. DAPI was used for nuclei staining of both the host and LD (indicated by white arrows). Result is representative of one of the four independent experiments. C. Splenic macrophages were isolated from uninfected (-LD) or LD infected BALB/c mice (+ LD) and HIF-1α was detected by indirect immunofluorescence assay using HIF-1α antibody. DAPI was used for detection of nuclei of both host and LD (indicated by white arrows). D. Real-time RT-PCR was performed from total RNA isolated from splenic macrophages isolated from LD-infected or uninfected mice using specific primers for HIF-1 target genes like VEGF, PAI-1, GLUT-1. β-actin was determined as a control. Results are representative of three independent experiments in each of which total RNA was isolated from macrophages derived from spleen of three mice.