Fig. 2

STAT3 phosphorylation and Endothelin-1 expression in the lung and macrophages from Calr^del10/WT mice. a Western blot of lung homogenates of the BMT recipients from Calr^WT/WT mice (WT-R) or Calr^del10/WT mice (del-R), immunoblotted with the indicated antibodies. b Phosphorylated STAT3 (p-STAT3) to total STAT3 (t-STAT3) or Endothelin-1 to β-actin ratios are shown in the graphs. The average value for WT-R under normoxia was set to 1 (n = 5, each). ^*P < 0.05 versus the corresponding normoxia group and ^†P < 0.05 versus the corresponding WT-R. c The lethally irradiated WT C57BL/6 J mice were transplanted with the BM cells from Calr^del10/WT/CAG-EGFP mice. These recipient mice were subjected to chronic hypoxia for 3 weeks, and then the lungs were fixed and stained with the indicated antibodies. Upper images show representative immunofluorescence of the lung sections stained with anti-GFP (green) and anti-αSMA (red) antibodies and DAPI (blue). Scale bars, 50 µm. Lower images show representative immunofluorescence of the lung sections stained with anti-GFP (green) and anti-F4/80 (red) antibodies and DAPI (blue). Scale bars, 10 µm. d-g BM mononuclear cells isolated from the Calr^WT/WT or Calr^del10/WT mice were cultured in the presence of 10 ng/mL of M-CSF for 6 days. d Representative immunofluorescence images of the cells stained with anti-F4/80 (green) and DAPI (blue) are shown. More than 90% of cells were macrophages expressing F4/80. Scale bars, 25 µm. e Dot plot of flow cytometry for cultured macrophages. Red, blue, and orange dots represent cells from Calr^WT/WT mice, Calr^del10/WT mice, and negative control (mixture of WT and Calr del10 cells), respectively. Over 90% WT and del10 cells were positive for both F4/80 and CD68. SSC indicates side scatter. f The cultured macrophages were then stimulated with 0.05 µg/mL of lipopolysaccharide (LPS), a potent activator of macrophages. The mRNA expression levels of Endothelin-1 (Edn1) were analyzed at the indicated time (n = 8, each). Actb was used for normalization. The average value for the macrophages from Calr^WT/WT mice at baseline was set to 1. g Left panels show western blots on STAT3, Endothelin-1, and β-actin in the macrophages stimulated with 0.05 µg/mL of LPS. Right graphs show phosphorylated STAT3 (p-STAT3) to total STAT3 (t-STAT3) or Endothelin-1 to β-actin ratios at the indicated time. The average value for the macrophages from Calr^WT/WT mice at the baseline was set to 1 (n = 4, each). All data are presented as means ± SEM. ^*P < 0.05 versus the corresponding WT group. WT, macrophages derived from the Calr^WT/WT mice; del10, macrophages from the Calr^del10/WT mice. Oligonucleotides and antibodies used are listed in Additional files 8, 9