Figure 5. Slc30a1 is required for iNOS/nitric oxide (NO) production and intracellular pathogen-killing capacity of macrophages in response to Salmonella infection.
(A) Time course of the intracellular pathogen-killing capacity of Salmonella-infected Slc30a1fl/fl;Lyz2-Cre and Slc30a1fl/fl bone marrow-derived macrophages (BMDMs) measured in colony-forming units per ml (n = 5). (B) Transmission electron microscopy images of Salmonella-infected BMDMs at 24 hpi. Red arrows indicate bacterial-containing phagosomes, and the insets show magnified images of bacterial engulfment. Scale bars, 5 µm. (C–E) RT-qPCR analysis of mRNAs encoding inflammatory cytokines (Tnfα, Il1β, and Il6), chemokines (Ccr2, Ccl2, Cxcl1, and Cxcl10) and macrophage biomarkers (Nos2 and Arg1) in BMDMs measured at the indicated times after Salmonella infection (multiplicity of infection [MOI] = 1) (n = 5). (F) Western blot analysis and summary of iNOS protein in Slc30a1fl/fl;Lyz2-Cre and Slc30a1fl/fl BMDMs either untreated or stimulated with Salmonella for 24 hr (n = 3). (G) Summary of nitrite concentration measured in the cell culture supernatant of BMDMs either untreated or stimulated with Salmonella for 24 hr (n = 5). (H) Western blot analysis and summary of p-p65 and p65 measured at the indicated times in Salmonella-infected BMDMs (n = 3). (I) RT-qPCR analysis of mRNA levels of genes involved in nuclear factor kappa B (NF-κB) signaling (p65, MyD88, Ikkα, and Ikkβ) measured in BMDMs 60 min after Salmonella infection (n = 5). (J) Western blot analysis and summary of p-p65 and p65 measured in uninfected and Salmonella-infected BMDMs either with or without N,N,N′,N′-tetrakis-(2-pyridyl-methyl)-ethylenediamine (TPEN) (4 µM) for 60 min (n = 4). (K) RT-qPCR analysis of p65, MyD88, Ikkα, and Ikkβ mRNA in uninfected and Salmonella-infected BMDMs either with or without TPEN for 60 min (n = 6). Data in this figure are represented as mean ± SEM. p values were determined using two-tailed unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, and ns, not significant.
Figure 5—figure supplement 1. Loss of Slc30a1 in the murine macrophages reduces iNOS and nitric oxide (NO) production by inhibiting the nuclear factor kappa B (NF-κB) pathway.
