Slit3, Robo1, and Robo4are expressed in vasculature. (A) Semiquantitative RT-PCR analysis determined the mRNA transcripts of Slit1-3 and Robo1-4 in HUVECs, primary mouse ECs from lung (LuEC), diaphragm (DiEC), and liver (LiEC), and primary rat brain VSMCs (BrSMC). (B). Real-time RT-PCR analyses of the expression profile of Slit1-3 (black bars) and Robo1-4 (red bars) in HUVECs. The data are presented relative to the Slit3 expression. (C-D). Coimmunostaining. Cryosections of adult mouse lung tissues were immunostained with anti-PECAM-1 (red), anti-α-SMA (red), and anti-His, anti-Slit3, anti-Robo1, or anti-Robo4 (all green) antibodies. Slit3, Robo1, and Robo4 colocalized with PECAM-1 (merged), whereas no anti-His staining was observed, showing that the staining of Slit3, Robo1, and Robo4 were specific. Cells outside PECAM-1–positive cells were observed to also express Slit3, Robo1, and Robo4 (indicated by white arrowhead), suggesting that Slit3 may also be expressed by VSMCs. This was confirmed by the colocalization of α-SMA with Slit3, Robo1, and Robo4 (merged) in vasculature. The colocalization of Slit3, Robo1, and Robo4 with PECAM-1 or α-SMA in the boxed areas in the merged images are shown at a higher magnification. High level of Slit3 and low levels of Robo1 and Robo4 are expressed by epithelial cells of bronchium (indicated by light blue arrowhead) but not bronchial SMCs (arrow), which express only low levels of Robo1 and Robo4. Scale bar, 120 μm.