Fig. 4.

HIF-1α-dependent up-regulation of iNOS after IFN-γ activation. (A and B) After BMDMs from WT mice or those lacking HIF-1α in their myeloid cell lineage (HIF-1 CKO) were infected with M. tuberculosis H37Rv (Mtb) for 12 h, extracellular bacilli were removed from the macrophage culture medium. BMDMs were cultured for 24 h with or without 500 U ml⁻¹ IFN-γ. Amplified iNos mRNA products were normalized to 18S rRNA. Data represent the means ± SD from four independent biological replicates (A). Whole-cell lysates were fractionated on 7.5% polyacrylamide gels with SDS–PAGE and immunoblotted with an anti-iNOS antibody or anti-β-actin antibody (as the loading control). The ratio of iNOS/β-actin with 500 U ml⁻¹ IFN-γ and M. tuberculosis was shown (B). Data represent the means ± SD from three independent biological replicates. (C and D) After BMDMs from WT mice or those lacking iNOS were infected with the kanamycin-resistant M. tuberculosis with luciferase (rMtb-luc) strain for 12 h, extracellular bacilli were removed from the macrophage culture medium. BMDMs were cultured for 4 days with or without 500 U ml⁻¹ IFN-γ (C), or in the absence or presence of 100 mM oxamate (Oxa), an LDH inhibitor (D). Intracellular M. tuberculosis growth was monitored by chemiluminescence (relative light units, RLU) with the luciferase assay system. Data represent the means ± SD from three independent biological replicates. The statistical significance of differences was assessed by a two-way ANOVA followed by the Bonferroni test (A), (C) and (D), or the Student’s t-test (B).