*4.10. Invasion Assay*

FluoroBlok™ Cell Culture Inserts with 8 µm pore size (Corning, New York, NY, USA; #351157) were coated using 0.8 mg/mL Matrigel (Sigma-Aldrich, St. Louis, Missouri, USA) and dried for 2 h at 37 ◦C and then at room temperature (RT) overnight. Matrigel was rehydrated using supplemented RPMI (Gibco, Thermo Scientific, Waltham, MA, USA; #11530586) [38]. Cells were harvested using 0.05 trypsin/EDTA (Gibco, Thermo Scientific, Waltham, MA, USA; #11580626), counted and reseeded at 2 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well, and kept under differentiating conditions (TS-Medium w/o CM, FGF4 and heparin) for five days. Medium in the lower chamber was changed every day. For analysis, Matrigel in upper chamber as well as medium in lower chamber were discarded and filters were washed several times with PBS. Filters were fixed and stained with 1% Hoechst (Sigma-Aldrich, St. Louis, MO, USA; #H6024) in methanol (VWR, Radnor, PA, USA; #20.847.307) for 10 min. After 3 washing steps with PBS, detection of invaded cells was carried out using a Leica DM LB microscope (Wetzlar, Hessen, Germany).

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/15/ 5503/s1. Figure S1: Spongiotrophoblast marker TPBPA reduced in KITD816V placentas, Figure S2: Validation of KITD816V-TSC, Table S1: Genotyping Primers, Table S2: Antibodies, Table S3: qRT-PCR Primers.

**Author Contributions:** Conceptualization, C.K., N.S., and H.S.; formal analysis, F.K., S.J., and D.N.; funding acquisition, H.S.; investigation, F.K., J.H., S.J., and D.N.; methodology, F.K., J.H., S.J., J.L., and H.S.; project administration, F.K., C.K., and N.S.; resources, N.P.; supervision, F.K., N.P., C.K., N.S., and H.S.; validation, F.K., J.K., D.N., and J.L.; writing—original draft, F.K. and H.S.; writing—review and editing, F.K., N.P., C.K., and H.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financially supported by DFG grant Scho 503 20/1 to H.S.

**Acknowledgments:** We kindly thank Angela Egert, Andrea Jäger, Gaby Beine, and Susanne Steiner for technical assistance. We also thank Marieta Toma and Laura Eßer for assisting with performance of Transwell invasion assays.

**Conflicts of Interest:** The authors declare no conflict of interest.
