Fig. 1
From: Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin
Characterisation of the in vitro mycobacterial infection of Gch1fl/fl Tie2cre macrophages. a Measurement of Nos2 and nitrite accumulation in bone marrow-derived macrophages stimulated with BCG (MOI 1:1) or IFNγ (10 ng/ml) and BCG (MOI 1:1) for 24 h in wildtype macrophages (n = 6). b Western blotting showing GTPCH and iNOS protein expression in Gch1fl/flTie2cre macrophages, compared to wildtype (WT) macrophages at baseline and following infection with IFNγ (10 ng/ml) and BCG (MOI 1:1) for 24 h. Equal protein loading was demonstrated by detection of β-tubulin. c Measurement of BH4 (pmol mg−1 protein) in bone marrow-derived macrophages (n = 4, p < 0.05). UT uninfected. d Nitrite accumulation in the cell culture media over 24 h of cell stimulation with BCG/IFNγ as before was measured using the Griess assay (n = 3/genotype). ND not detectable. e Quantification of the NMA-inhibitable arginine to citrulline conversion (n = 8, p < 0.05). f Representative EPR spectra from n = 4/genotype WT and Gch1fl/flTie2cre macrophages following activation with IFNγ (10 ng/ml) and BCG (MOI 1:1). g Cells were incubated with dihydroethidium for the last 30 min of culture and ethidium production was measured in cell lysates by HPLC (n = 3/genotype). h Flow cytometry dotplot demonstrating identification of neutrophils (7/4HI, Ly-6GHI) and inflammatory monocytes (7/4HI, Ly-6G−) in lavage fluid from wildtype animals 6 h after injection with 1 mg zymosan ip. i The recruitment of monocytes and neutrophils by zymosan or saline control injection into wildtype and Gchfl/flTie2cre animals was quantified by flow cytometry in the peritoneal lavage 6 h later (n = 6 control groups, n = 5 Gch1fl/fl Zymosan, n = 6 Gch1fl/flTie2cre Zymosan). Lavage fluid was further analysed for total nitrite and nitrate (NOx) content by NO analyzer (j) and total biopterin content (k) (n = 5 Gch1fl/fl Control, n = 2 Gch1fl/flTie2cre Control, n = 5 Gch1fl/fl Zymosan, n = 6 Gch1fl/flTie2cre Zymosan, p < 0.05). l The elicited peritoneal cells were assayed for BH4 content (n = 4 per group, two-tailed t-test, p < 0.05). All data analysed by one-way or two-way (genotype experiments) ANOVA with Bonferroni post-test unless otherwise stated above. All ‘n’ represent individual biological replicates, including cell cultures from individual animals/genotype. Data shown as individual data-points with mean and SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Source blot and flow cytometry data are available in Supplementary Figure 10 and 11
