FIGURE 4.

MRTF silencing in decidual fibroblasts reduces actomyosin contractility and resistance to trophoblast invasion. (A) Immunofluorescence of dESFs with CRISPR/Cas9 based gene silencing for scrambled control, and MKL1 & MKL2 (MKL1/2^KO) showing F-actin and myosin light chain-9 (MYL9), quantification of intensity per cell for MYL9 shows no statistical difference (B); scale bar = 25 µm. (C) Immunofluorescence images showing reduced abundance of phospho-MYL9 in MKL1/2^KO dESFs (D); scale bar = 20 µm. (E) Relative expression of myosin light chain kinase (MYLK) hESF, dESF, as well as dESFs co-cultured or conditioned with HTR8. (F) Pearson’s coefficient of phospho-MYL9 (green) co-localization with F-actin fibers (red) in Scrambled control, and MKL1/2^KO dESFs. (G) Schematic showing the setting of the ANSIA assay to measure stromal invasion of H2B-mCherry expressing HTR8s in the dESF monolayer on a nanopatterned substrate with the anisotropic nanoridges aligned orthogonal to the patterned HTR8-dESF interface. (H) Representative time-stamped images of HTR8 (red)-dESF (unlabeled) interface at 0 and 24 h, with dESFs either silenced with scrambled gRNA, or gRNA targeted towards MKL1. (I) Extent of stromal invasion by H2B-mCherry labeled HTR8 cells into the dESF monolayer for conditions in e, and significance is calculated using Anova followed by Tukey’s test; n > 10 per condition.