Fig. 4
Epithelial derived CXCL10 activates endothelial cells and allows myeloid cells trans-endothelial migration through a CXCR3 / Src kinase dependent signaling. (A) Calu-3 cells were seeded at the bottom of the transwell chamber for 48 h before they were cocultured with confluent HUVEC endothelial cells placed in the upper chamber, together with a THP1 monocytes suspension for 24 h. (B) Pictures of activated THP-1 cells which have transmigrate through HUVEC confluent monolayer. The number of transmigrating cells was then counted. (C) Quantification of (B). The results are presented as the means ± SDs (one-way ANOVA; *P < 0.05, ***P < 0.001). (D) Western blot analysis of p-Src, Src, p-ERK and the ERK protein. Vinculin was used as a loading control. (E) Quantification of the data in (D). Bar graphs are presented as the means ± SDs. (F) THP-1 cells were placed in the upper chamber, and migration was stimulated by CXCL10 in the lower chamber. The number of transmigrating cells was then counted after DAPI staining. Treatment with 1 µg/ml CXCL10 alone (blue) or in combination with 6 nM SCH546738 or 1 nM PP2 compared to the control. (G) ELISA dosage of CXCL10 in the supernatant of HUVECs, control scrambled Calu-3 cells, Calu-3 cells transfected with CXCL10 expressing lentivirus alone or in coculture with HUVECs. The results are presented as the means ± SDs (one-way ANOVA; *P < 0.05, **P < 0.01, and ***P < 0.001).
