Pf670462

Doxorubicin (cat#D1515), hydroxyurea (cat#H8627), DAPI ([4’, 6-diamidino-2-phenylindole]dihydrochloride, cat# D9542), cycloheximide (cat#C-7698) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PF670462 (cat#3316) and LH846 (cat#4896) were purchased from TOCRIS (Bristol, BS11 0QL, United Kingdom). Propidium iodide was from Life Technologies (cat#P3566).

Trophoblast cells derived from a 6–12 weeks-old human placenta (HTR-8 SVNeo, referred to as HTR-8 cells, ATCC #CRL-3271) were cultured in RPMI 1640 Medium (Gibco, #11875093) with 10% FBS (Sigma-Aldrich, #F4135) and 1% Penicillin–Streptomycin (Sigma-Aldrich Cat# P4333). HTR-8 cells were plated at 0.5 million cells/ml per well in a 24-well wound healing assay (Abcam, #ab242285). Twenty-four h after plating, the HTR-8 cells formed a monolayer and the inserts were removed, where after the cells were treated with vehicle (DMSO 1/500 or 1/5000 dilution, no difference in HTR-8 cell migration in response to DMSO concentration was found and data were pooled), KL001 (Tocris Bioscience, # 46-851-0), PF670462 (Tocris Bioscience™ Cat# 33-161-0), SR9009 (Tocris Bioscience, # 5855), SR8278 (Tocris Bioscience, # 4463), or KL101 (Sigma Aldrich, #SML3014) at 1 μM and 10 μM. Bright field image acquisition of the wound healing area was done using a light microscope (Leica DMi1) 0, 24 and 48 h after treatment. Data were analyzed using ImageJ/Fiji^® version 1.53 (NIH).

IC261 was from Calbiochem, LaJolla, CA, USA. PF-670462 was obtained from Tocris bioscience, Bristol, UK. TG003 and TG001 were synthesized according the procedures described previously
[13 (link)]. These drugs were made up as concentrated stock solution in DMSO, aliquoted and stored at –20°C. An aliquot was diluted to the desired concentration in saline or Krebs solution immediately prior to use. The dose ranges of IC261 and TG003 used were determined according to our previous report (for IC261)
[12 (link)] and preliminary study (for TG003).