Figure 7
TGFβ1 and BMP2 are expressed in circulating erythrocytes. (a) Representative image showing TGFβ1 expression in PcNobs (n = 6). PcNobs in blood cell suspensions were double labeled with TGFβ1 (green) and Newtic1 (red) antibodies. Nuclei were also labeled with Topro-3 (red). Arrowheads point to Newtic1-positive (Newtic1+) PcNobs. PcNobs exhibited TGFβ1 immunoreactivity but their fluorescence intensity was variable. Note that the fluorescence intensity was sometimes higher along the equator of PcNobs, independent of the intensity of Newtic1 immunoreactivity. Scale bar, 100 μm. Right hand column shows a particular Newtic1+ PcNob exhibiting intense TGFβ1 immunoreactivity in its cytoplasm. Upper two panels show confocal images. Scale bar, 20 μm. (b) Representative image showing BMP2 expression in PcNobs (n = 3). PcNobs in blood cell suspensions were double labeled with BMP2 (green) and Newtic1 (red) antibodies. The data are presented in the same manner as in (a). PcNobs exhibited BMP2 immunoreactivity, but as in TGFβ1, their fluorescence intensity varied. (c) Control immunoreactivity (n = 3). PcNobs in blood cell suspensions were labeled with control (RFP; green) antibody. The data are presented in the same manner as in (a). (d) Relationships of TGFβ1 expression in PcNobs with either their developmental stage or Newtic1 expression. We used representative images obtained from six experiments in (a). Total number of PcNobs at an early stage: Newtic1−, n = 51; Newtic1+, n = 41. Total number of PcNobs at an intermediate/late stage: Newtic1−, n = 255; Newtic1+, n = 110. (e) Relationships of BMP2 expression in PcNobs with either their developmental stage or Newtic1 expression. We used representative images obtained from three experiments in (b). Total number of PcNobs at an early stage: Newtic1-, n = 74; Newtic1+, n = 11. Total number of PcNobs at an intermediate/late stage: Newtic1−, n = 231; Newtic1+, n = 30. Statistical difference: Student’s t-test/Welch’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ND (no difference) (see Methods).
