*3.2. F-Actin Organization and Focal Adhesion Formation Are Affected by Simulated ECM Stiffness in JAR Cells*

Since cell spreading is regulated by the cytoskeleton and focal adhesion complex [43], we measured the assembly of F-actin and the focal adhesion in JAR cells on simulated ECM with different stiffnesses (Figure 2A). The images revealed significant differences in actin organization and focal adhesion formation between three simulated ECM regions with different stiffnesses. The focal adhesion area indicated by the staining of vinculin, which was measured by ImageJ software, increased as simulated ECM stiffness increased (Figure 2B). To quantify the F-actin cytoskeleton structure differences, we applied a steerable filter to extract F-actin bundles [32] (Figure 2C). The results demonstrated that JAR cells in the stiff simulated ECM region had longer and more robust stress fibers compared to other simulated ECM regions (Figure 2D). This demonstrates that stiff simulated ECM enhanced the F-actin organization and focal adhesion assembly of JAR cells.

**Figure 1.** Simulated ECM stiffness regulates trophoblast cell morphology and spreading area. (**A**) (**Top**): schematic diagram of PA gel with stiffness gradient. The Young's modulus of the PA gel is 1 kPa, and its apparent Young's modulus gradually increases with the decrease in the gel thickness. According to the change of the apparent Young's modulus, the surface of the PA gel is evenly divided into three regions, namely Stiff, Intermediate, and Soft. (**Bottom**): schematic representation of JAR cells cultured on different regions. E indicates the apparent Young's modulus. (**B**) Phase contrast images of JAR cells cultures on different regions scale bar: 20 µm. The yellow dashed line indicates the boundary of cells. (**C**) Measured average cell spreading area of JAR cells cultured on different regions. \*\*\*\* *p* < 1 × 10 −6 , *n* = 31, 27, 23 for Soft, Inter and Stiff, respectively. Data reported as mean ± standard deviation for *N* = 3 independent experiments. Each scatter indicates each cell being measured, and each color indicates an independent experiment.

#### *3.3. Stiff Simulated ECM Enhances JAR Cell Motility*

Several studies demonstrated that cell migration is regulated by ECM stiffness [44–48]. To investigate cell migration behavior on ECM with different stiffness, we used a time-lapse microscope to visualize the motility of JAR cells. To examine the relationship between ECM stiffness and JAR cell migration, we used particle image velocity (PIV) to analyze the movement of JAR cells on simulated ECM with different stiffnesses (Figure 3A). Meanwhile, we selected JAR cells in different simulated ECM regions (stiff, intermediate, and soft) and tracked their migration for 5 h (Figure 3B). The migration distance (track length) as well as the migration velocity (track\_length/time) of JAR cells on the inter and stiff regions were significantly increased compared to JAR cells on the soft region (Figure 3C,D). The displacement of JAR cells significantly increased as simulated ECM stiffness increased (Figure 3E). These results indicate that stiff simulated ECM increases cell motility in JAR cells.

**Figure 2.** F-actin organization and focal adhesion formation are affected by simulated ECM stiffness in JAR cells. (**A**) Immunofluorescence staining of JAR cells cultured on different regions (red: F-actin; green: vinculin, scale bar: 20 µm). (**B**) Measured focal adhesion area of JAR cells cultured on different regions. \*\*\*\* 1 × 10 −6 , *n* = 18, 27, 24 for Soft, Inter, and Stiff, respectively. Data reported as mean ± standard deviation for *N* = 3 independent experiments. Each scatter indicates each focal adhesion being measured. (**C**) Skeletonization of F-actin in JAR cells cultured on different regions (scale bar: 20 µm). (**D**) Measured cytoskeleton length of JAR cells cultured on different regions. \*\*\*\* 1 × 10 −6 , *n* = 35, 27, 24 for Soft, Inter, and Stiff, respectively. Data reported as mean ± standard deviation for *N* = 3 independent experiments. Each scatter indicated each F-actin filament being measured.

**Figure 3.** Stiff simulated ECM enhances JAR cell motility (**A**) (**Top**): Vectors of JAR cell migration. (**Bottom**): heatmap of velocity magnitude on different regions of simulated ECM (scale bar: 50 µm, color bar: 0~0.2 µm/min). (**B**) Tracking of JAR cells cultured on different regions (scale bar: 100 µm, time bar: 5 h). Each scatter indicated each cell being analyzed. (**C**,**D**) Velocity and track length of JAR cell migration. \*\*\*\* *p* < 1 × 10 −6 , \*\*\* *p* < 0.001, *n* = 11, 11, 9 for Soft, Inter, and Stiff, respectively. Data reported as mean ± standard deviation for *N* = 3 independent experiments. Each scatter indicated each cell being analyzed. (**E**) Track displacement of JAR cells cultured on different regions. \*\*\* *p* < 0.001, \*\* *p* < 0.01, *n* = 11, 11, 9 for Soft, Inter, and Stiff, respectively. Data reported as mean ± standard deviation for *N* = 3 independent experiments.
