Sipak1

EnSCs were subjected to treatment with PlGF (#P1588, Sigma) at a concentration of 20 ng/ml for 6 days^{68 (link)}. For decidualization, EnSCs were treated with 0.5 μM 8-Bromoadenosine-3’,5’-cyclic monophosphate sodium salt (cAMP) (#1140, Tocris) and 1 μM Medroxyprogesterone 17-acetate (MPA) (#M6013, Sigma) for 6 days^{69 (link)}. The cell culture medium was replaced every 48 h with fresh treatment media. The experimental groups are indicated as control (untreated EnSCs), PlGF, cAMP+MPA, and cAMP+MPA+ PlGF.
Where indicated, EnSCs were treated with pravastatin (Pravastatin sodium salt hydrate, #P4498, Sigma) with or without PlGF treatment at 10 µM for 24 h^{70 (link)}. The experimental groups are classified as control (untreated EnSCs), PlGF, pravastatin (Prav), and pravastatin + PlGF (Prav + PlGF). Epidermal Growth factor (EGF) was used as a positive control for Rac1 activation. (#324831, Sigma) at a concentration of 100 ng/ml for 24 h^{71 (link)}.
For gene silencing, EnSCs were treated with siRNAs^{72 (link)}, such as siRac1 (50 nM, #L-003560-00-0010, Dharmacon), siPAK1 (20 µM, #L-003521-00-0010, Dharmacon), and siWAVE2 (5 nM, #s55787, ThermoFisher Scientific). The siRNAs were transfected with Lipofectamine RNAiMAX (#13778075, ThermoFisher Scientific) for 48 h with and without combination with PlGF treatment.

The 24ST1NLESG cells were transfected by electroporation using the Neon Transfection System (Life Technologies, Carlsbad, CA, USA). Electroporation parameters were 1350 Pulse Voltage (V), 10 Pulse Width (ms), 3 Pulse Number, 2 × 10⁷ cell/mL. Final concentrations were 1 × 10⁷ cell/mL, 1 µM siRNA (siPAK1, Dharmacon, cat # L-003521-00-0020; siPAK2, Dharmacon, cat # L-003597-00-0020; siPAK4, Dharmacon, cat # L-003615-00-0020; siMAPK1, Thermofisher cat # 4390824; siPKA, Thermofisher cat # 4390825; Non-targeting Control, Dharmacon, cat # D-001810-10-20). The efficiency of siRNA knockdown was assessed by Western blot analysis. Anti-PAK1 (cat # 2602S), anti-PAK2 (cat #2608S), anti-PAK4 (cat # 62690), anti-PKA (cat # 4782), anti-MAPK (cat # 9108), and anti-GAPDH (cat # 97166S) antibodies were purchased from Cell Signaling. The efficiency of siRNA knockdown was quantified using ImageJ.