Fig. 5

Decreased BMPR2 is linked to decreased CD14 expression. (A) RT-qPCR of BMPR2 gene expression in classical monocytes of IPAH patient. (B) RT-qPCR of CD14 gene expression in classical monocytes of IPAH patients. (C) RT-qPCR of CD14 gene expression in THP1 monocyte cell line, where BMPR2 was decreased via treatment with siRNA targeting BMPR2 (siBMPR2) vs. non-targeting siRNA (siControl). (D-G) RT-qPCR of STAT1, IFN-α, IFN-β and IFN-γ gene expression in THP1 monocyte cell line, where BMPR2 was decreased via treatment with shRNA targeting BMPR2 (shBMPR2) vs. non-targeting shRNA (shControl) (H) Schematic outlining the breeding strategy used to generate mice with tomato (Td) fluorescent monocyte-specific tag and a conditional, monocyte specific Bmpr2 deletion (MON-Bmpr2^−/−, Cx3cr1Td Bmpr2^−/−), and Control mice (Control; Cx3cr1Td). Details are provided in the Methods. (I) RT-qPCR of Ly6c (mouse CD14) gene expression in circulating monocytes of the MON-Bmpr2^−/− mouse, compared to Control. n = 3 groups, each consisting of pooled blood from 3 mice. (J) LY6C protein in MON-Bmpr2^−/− bone marrow derived macrophages (mBMDM) assessed via western blot, with representative blot image. (K) Representative microscopic images of mBMDM stained with CD68 (green), DAPI (blue) and tamoxifen induced Td (red). MON-Bmpr2^−/− mBMDM were consistently smaller compared to Control. Scale bar = 50 μm, 20 μm (zoom). (L) Representative microscopy images with small pulmonary artery (white dashed outline) zoom insert, of lung sections of mice, labeled with DAPI (blue) and showing Td (red) labelled macrophages following hypoxia. Scale bar = 100 μm, 50 μm (zoom). With quantification of macrophages per field of view (FOV). Bars represent mean ± SEM. *p < 0.05, **p < 0.01, by Mann-Whitney non-parametric t-test (A, B, J). For E, F, H, I, n = 3 biological replicates; + denotes the minimum achievable P-value for n = 3 was reached by the non-parametric t test