Fig. 4

Neutrophils, but not macrophages, mediate EMT related gene expression in HRas^V12 expressing epithelial cells.

(A–C) Fluorescent Z stack projections of live 3.5 dpf transgenic mpx:GFP (green neutrophils) control MO injected (A), Rac2^D57N (B) and irf8 MO (C) larvae expressing HRas^V12. (D–E) Quantification of neutrophil recruitment (as a ratio of neutrophils per transformed cell) shows a significant decrease in neutrophil recruitment to HRas^V12 expressing cells in Rac^D57N embryos when compared to controls (D), no significant change was observed in neutrophil recruitment in irf8 morphant larvae compared to control (E). (F–H) Fluorescent Z stack projections of live 3.5 dpf of transgenic mpeg:Dendra (green macrophages) control MO injected (F), Rac2^D57N (G) and irf8 MO (H) larvae expressing HRas^V12. (I–J) Quantification of macrophage recruitment (as a ratio of macrophages per transformed cell) shows a significant decrease in macrophage recruitment to HRas^V12 expressing cells in irf8 morphants compared to controls (D). No significant change was observed in macrophage recruitment in Rac2^D57N larvae compared to control (E). (K) Quantitative RT-PCR (one representative graph shown n = 4) indicates a statistically significant decrease in mmp9 and slug transcripts in transformed cells from Rac2^D57N larvae compared to control MO injected larvae while no significant decrease was seen in mmp9 and slug transcripts in transformed cells from irf8 Mo injected larvae compared to controls. (L–M) Double label WMISH with HRas^WT (A) and HRas^V12 (B) transcript labeled in red and cxcl8 transcript label in blue. cxcl8 expression is induced in HRas^V12 expressing larvae compared to control HRas^WT expressing larvae. *** = P<.001, **** = P<.0001, ns = not significant. Scale bar = 20 microns.