Human trophoblast cell line HTR8/SVneo was maintained as described25 (link). Additional human trophoblast cell line Sw.71 was kindly provided by Dr. Gil Mor (Yale University), and maintained as described previously29 (link). In siRNA experiments, cells were transfected with the siRNAs (ThermoFisher) shown in Online Supplemental Materials and Methods using siPORT Amine transfection reagent as previously described24 (link). Control siRNA was purchased from GE Dharmacon (ON-TARGETplus Non-targeting Control siRNA). For reconstitution experiments, cells were first transfected with siRNA targeting endogenous ApoER2, and subsequently the cells were transfected with adenoviral particles (1010 particles/ml) encoding ApoER2 constructs24 (link). Twenty-four hours after adenoviral transfection, experiments were performed. Pharmacological inhibition studies were performed using HIF1α inhibitor (1μM, GN44028, TOCRIS), MMP14 inhibitor (10nM, NSC405020, Sigma-Aldrich), or p38 inhibitor SB 202190 (10 μM, Selleckchem) in the presence of NHIgG or aPL (100 μg/ml).
Nsc405020
Manufactured by Merck Group
Sourced in Germany
NSC405020 is a laboratory product manufactured by Merck Group. It is a specialized piece of equipment designed for use in research and scientific applications. The core function of NSC405020 is to facilitate specific laboratory procedures and experiments. However, a more detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation on its intended use.
Automatically generated - may contain errors
3 protocols using nsc405020
1
Trophoblast Cell Line Maintenance and Manipulation
2
Inhibition Studies of MT1-MMP and Tideglusib
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For inhibition studies, the following compounds were used: MT1-MMP inhibitor, NSC405020 (444295 Sigma-Aldrich, Darmstadt, Germany) in 0.1% DMEM, tideglusib SML0339 (Sigma-Aldrich, Darmstadt, Germany). In all cases, compounds were dissolved in DMSO. MLE-12 cells or PCLS were incubated in the presence of the inhibitor or vehicle only in the case of controls.
3
Spheroid Formation and Cell Migration
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Cells were seeded into micro-molds to form spheroids for 72 hours as explained above. Three different conditions were used: first, media were not changed; second media were changed daily and it contained either 50 µg/mL ascorbic acid; or contained 1 mM hydroxyurea as third option. Thereafter spheroids were transferred from molds to 96-well plate coated with Collagen I 5 µg/cm 2 o/n at +4°C and blocked with 0.1% bovine serum albumin (BSA)-PBS 1 hour at +37°C. In migration study KSFM was added on top of the spheroid. In invasion study Collagen I gel was first added on top of the spheroid and KSFM was added into the wells after gel was properly formed.
Spheroids were imaged every 24 hours for 4 days by using IncuCyte ZOOM System. To study the effects of MMP inhibition on the cells invasion medium was supplemented with 1 µM NNGH (Sigma) or 10 µM NSC 405020 (Sigma) with respectively amount of DMSO as control.
Spheroids were imaged every 24 hours for 4 days by using IncuCyte ZOOM System. To study the effects of MMP inhibition on the cells invasion medium was supplemented with 1 µM NNGH (Sigma) or 10 µM NSC 405020 (Sigma) with respectively amount of DMSO as control.
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