*2.7. Signaling Cascades and Invasion Capability are A*ff*ected in KITD816V-TSC*

Previously, we had shown that, in the hematopoietic system, expression of KITD816V leads to a block of differentiation of erythroblasts and a continuation of proliferation of precursor cells. There, we had demonstrated that KITD816V leads to induction of MAPK signaling as well as diminished AKT activation [27]. Hence, we used KITD816V-TSC to examine the activation of key signaling pathways during 9 days of TSC differentiation (Figure 5C). On day 0, levels of phosphorylated (P-) ERK and P-AKT in KITD816V TSC appeared reduced and comparable to day 3/6 of differentiating WT-TSC. Of note, on day 0, levels of P-STAT3 are comparable but become upregulated over the course of

differentiation in KITD816V-TSC (Figure 5C). This suggests that chronic activation of the KIT signaling cascade in KITD816V-TSC results in diminished levels of P-ERK and P-AKT, priming the cells for rapid and premature differentiation. *Int. J. Mol. Sci.* **2020**, *21*, x 10 of 18

**Figure 5.** Differentiation of KITD816V-TSC: (**A**) Photomicrographs depicting *in vitro* differentiation of KITD816V-TSC line #3 and WT-TSC line 2.1 cultured for 0, 3, 6, and 9 days under differentiation conditions. White arrows indicate TGCs. Scale bar represents 250 μm. (**B**) qRT-PCR analysis of endogenous expression of *Pl1*, *Pl2*, and *Plf* in KITD816V-TSC line #3 and WT-TSC line 2.1 in undifferentiated states and after culture under differentiation conditions for 6 days. RNA was obtained from three biological replicates; expression was normalized to the housekeeping gene *Gapdh.* Data is represented by mean value ± SD. Significance was determined by unpaired t-test and indicated with \**p* < 0.05. (**C**) Western Blot detected phosphorylated Extracellular-Signal Regulated Kinase (ERK), Protein Kinase B (AKT), and Signal Transducers and Activators of Transcription 3 (STAT3) during differentiation of KITD816V-TSC line #3 and WT-TSC line 2.4 in comparison to β-ACTIN. (**D**) Representative photomicrographs of Hoechst-stained nuclei of cells that invaded through Matrigel coated filter membranes after five days under differentiating conditions: White arrows indicate nuclei of TGCs. Scale bar represents 250 μm. (**E**) Quantification of invaded cells per filter in KITD816V-TSC line and WT-TSC line (biological replicates = 2): Data is represented by mean value ± SD. Significance was determined by unpaired t-test and indicated with \* *p* < 0.05. **Figure 5.** Differentiation of KITD816V-TSC: (**A**) Photomicrographs depicting in vitro differentiation of KITD816V-TSC line #3 and WT-TSC line 2.1 cultured for 0, 3, 6, and 9 days under differentiation conditions. White arrows indicate TGCs. Scale bar represents 250 µm. (**B**) qRT-PCR analysis of endogenous expression of *Pl1*, *Pl2*, and *Plf* in KITD816V-TSC line #3 and WT-TSC line 2.1 in undifferentiated states and after culture under differentiation conditions for 6 days. RNA was obtained from three biological replicates; expression was normalized to the housekeeping gene *Gapdh.* Data is represented by mean value ± SD. Significance was determined by unpaired t-test and indicated with \* *p* < 0.05. (**C**) Western Blot detected phosphorylated Extracellular-Signal Regulated Kinase (ERK), Protein Kinase B (AKT), and Signal Transducers and Activators of Transcription 3 (STAT3) during differentiation of KITD816V-TSC line #3 and WT-TSC line 2.4 in comparison to β-ACTIN. (**D**) Representative photomicrographs of Hoechst-stained nuclei of cells that invaded through Matrigel coated filter membranes after five days under differentiating conditions: White arrows indicate nuclei of TGCs. Scale bar represents 250 µm. (**E**) Quantification of invaded cells per filter in KITD816V-TSC line and WT-TSC line (biological replicates = 2): Data is represented by mean value ± SD. Significance was determined by unpaired t-test and indicated with \* *p* < 0.05.

Among their various functions such as guiding the attachment of the blastocyst and secretion of essential hormones and proteins, TGCs are also capable of invading into uterine tissue to establish the vital connection to the maternal blood vessels [31,37]. Finally, we analyzed the invasive capacity of KITD816V-TGCs in comparison to WT-TGCs by using a Transwell migration assay. Previously, it was shown that higher Matrigel concentrations result in preselection of TGCs as non-TGCs do not invade through thicker Matrigel layers [38]. Also, lower cell densities led to increased invasion [38]. Hence, we seeded 2 <sup>×</sup> <sup>10</sup><sup>4</sup> cells on a layer of 0.8 mg/mL Matrigel and omitted FGF4, heparin, and CM from culture medium to induce differentiation. After five days, Hoechst staining showed the presence of nuclei resembling TGCs in size and structure, indicating successful invasion (Figure 5D). Quantification of two independent experiments revealed that a significantly higher number of KITD816V-TGCs had migrated through the Matrigel and the pores (Figure 5E). Thus, KITD816V signaling in trophoblast cells seems to result in a higher portion of invasive cells. This might be due to the fact that KITD816V-TSC are much faster in inducing differentiation and gain migratory capabilities earlier than WT-TSC.
