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Fig. 5. Transmigration of HTR-8/SVneo through a stromal cell layer. (A) DiI-labeled DSC were plated on the upper side of the filters and allowed to attach. Their spontaneous migration was evaluated before (left side) or after (right side) the addition of unstained HTR-8/SVneo cells. The migration was stopped after 5 hours and the filters were stained with the nuclear dye Hoechst 33342. DSC did not migrate at all (note the absence of red color on the lower part of the filters). The white arrows indicate the nuclei of migrated HTR-8/SVneo cells. Bar, 75 µm. (B) DSC were plated on the lower side of an inverted FluoBlok insert and incubated for 72 hours. The insert was then placed in the chamber and HTR-8/SVneo cells labeled with DiI were added on the upper side. The chambers for migration are schematically represented in the figure. Quantification of HTR-8/SVneo cells migrated through DSC grown on the upper or lower side of the filter is reported in the graph. When DSC were grown on the lower side of the filter, HTR-8/SVneo cell migration was significantly higher than that observed with DSC monolayer in the upper side of the insert. Bar, 75 µm. (C) Inhibition of HTR-8/SVneo and EVT cell transmigration. In some instances, anti-gC1q1 or anti-
4 integrin subunit antibodies were added alone or together to block cell transmigration with DSC monolayer on the lower side of the filter. Anti-1 integrin subunit antibody was used as negative inhibition control. Data are expressed as mean ± s.d. of four experiments under baseline conditions (mAb anti-1). Post-hoc comparison of mAb anti-1 versus specific antibodies after significant ANOVA for repeated-measures main effect, Dunnet's test. *P<0.05, **P<0.01. (D) HTR-8/SVneo cell transmigration under EMILIN1 gene silencing conditions. Upper panel: decidual stromal cell EMILIN1 mRNA levels (30 amplification cycles) at 48, 72 and 96 hours after transfection with the siRNA-specific SMARTpool. Lane 1: control, lipofectamine-treated cells; lane 2: scrambled siRNA; lane 3: EMILIN1 siRNA. RT-PCR analysis reveals that the levels of EMILIN1 mRNA decrease only at 48 hours. Lower panel: evaluation of HTR-8/SVneo cell transmigration after the EMILIN1 gene silencing in decidual stromal cell. The three confocal images, concerning different fields of FluoroBlok inserts, represent the staining of EMILIN1 (in green) produced by decidual stromal cells treated with lipofectamine, transfected with scrambled siRNA or with EMILIN1 siRNA and cultured on the lower side of a FluoBlok filter for 48 hours. Fast DiI-labeled HTR-8/SVneo (in red) were fixed after 7 hours of migration. Bars, 100 µm.
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