Fig. 1.

Expression of HIF-1α in TB granulomas and macrophages. (A) The lungs of each of seven BALB/c mice 5 weeks after infection with or without M. tuberculosis Erdman were fixed in 10% formaldehyde. A normal mouse lung is shown in i–iv and mouse lung granuloma in v–viii. The mouse lung sections were stained with Hematoxylin-Eosin stain (i, v) or the Ziehl–Neelsen stain (ii, vi), and immune-stained with anti-HIF-1α IgG (iii, vii) and anti-MDP1 IgG (iv, viii). Scale bars: 100 μm (i–viii). (B and C) After infection with M. tuberculosis H37Rv (Mtb) or M. bovis BCG (BCG) for 12 h, BMDMs from WT mice were cultured for 24 h in the absence or presence of 500 U ml⁻¹ IFN-γ. (B) Whole-cell lysates were fractionated on 7.5% polyacrylamide gels with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an anti-HIF-1α antibody, anti-HIF-1β antibody or anti-β-actin antibody (as the loading control). Ratios of HIF-1α/β-actin are shown as relative intensities. Values are expressed as means ± SD from four independent biological replicates. (C) Amplified Hif-1α mRNA products were normalized to 18S rRNA. Values are expressed as the means ± SD from three independent biological replicates. (D) After infection with M. tuberculosis H37Rv for 12 h, BMDMs from WT mice or those lacking Toll-like receptor 2/4/9 (TLR2/4/9) were cultured for 24 h without 500 U ml⁻¹ IFN-γ. Whole-cell lysates were fractionated on 7.5% polyacrylamide gels with SDS–PAGE and immunoblotted with an anti-HIF-1α antibody or anti-β-actin antibody (as the loading control). The significance of differences was assessed by a two-way analysis of variance (ANOVA) followed by the Bonferroni test.