HER1 Signaling Mediates Extravillous Trophoblast Differentiation in Humans: JAR Cell Migration Assays

HER1 Signaling Mediates Extravillous Trophoblast Differentiation in Humans: JAR Cell Migration AssaysTreatment of Cell Lines and Fluorescent Immunocytochemistry
After a period of 24 h of serum starvation (above), JAR cells were preincubated with either vehicle (0.0025% DMSO) or AG1478 (2 iM in 0.0025% DMSO) for 30 min. JAR cells were then stimulated with either DCM + vehicle, EGF + vehicle, or +/- AG1478 (2 iM in 0.0025% DMSO) as indicated in each experiment for 24 h. DCM was collected from primary first trimester decidual cell cultures as previously described. Briefly, decidual cells were serum-starved in 0.2% BSA (Sigma) RPMI supplemented with Normocin (serum-free DCM) for 48 h. Following the treatment period, immunocytochemistry was performed on confluent JAR monolayers. JAR cells were fixed with 4% paraformaldehye, permeabilized using 0.02% triton X100, and quickly exposed to Sudan Black. All primary and secondary antibodies used in these procedures are detailed in Table 1.
Primary and secondary antibodies were prepared and used as described above. Slides were coverslipped using 90% glycerol. All incubations were performed in a light-protected incubation chamber. Fluorescent images were captured as described above.
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JAR Cell Migration Assays
JAR choriocarcinoma cells were seeded in fibronectin-coated 8-im pore transwell cell culture inserts (100 000 cells per well in SFM; Becton Dickinson) and allowed to attach for 8 h under the culture conditions described above. The protocol for migration experiments involving the HER1 antagonist (2 iM AG1478), HER2 antagonist (5 iM AG825), or HER1 signaling pathway inhibitors (1 iM PD90589, 5 iM SB203580, 100 or 50 nM Ro318220, or 10 or 1 iM LY294002; CedarLane) involved a 30-min pretreatment of JAR cells with the antagonist/inhibitor prior to placing the JAR cell-containing inserts into culture wells containing HBEGF (Sigma)-supplemented SFM. Serum-free vehicle controls were also performed by the addition of 0.0025% DMSO. After 48 h of exposure to HBEGF, migrated JAR cells were fixed, stained with Diff-Quik (Dade Behring, Milton Keynes, U.K.), and mounted in 90% glycerol and 10% PBS. Four random fields, each 1 cm2 per filter, were counted at 200X magnification to obtain a mean value for each filter. Each experiment was carried out in triplicate and performed as four independent experiments.

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