Nsc23766

Inhibitors of c-Raf (Kobe2602), Ras (FTaseII), Rac (NSC 23766), and Gβγ (gallein) were purchased from Cayman Chemical Co. Inhibitors of cAMP (SQ22536) and PKA (H89), bafilomycin A1, caveolin (genistein), the selective Gα inhibitor suramin, the PI3K inhibitor wortmannin, and GDP were purchased from Sigma-Aldrich. Anatag3 (ML-224) was obtained from DC Chemicals. Inhibitors of ERK1/2 (PD98059), PKC (Go6983), and Rho (Y-27632) and the nonselective PKC-δ inhibitor rottlerin were purchased from Calbiochem/EMD Millipore. The dynamin inhibitor dynasore, the Gβγ inhibitor hydrobromide, and the endocy-tosis inhibitor PAO were from Tocris Bioscience. The clathrin inhibitor Pitstop was from R&D Systems Inc. The Gα_q/11 inhibitor YM-254890 was obtained from Wako Chemicals USA Inc.

Inhibitors were added to cells prior to imaging at the following concentrations and timepoints: Cdc42 inhibitor ZCL 278 (Cayman Chemical, 14849), 7.5 μM, 2 hours prior to imaging; Rac1 GEF inhibitor NSC 23766 (Cayman Chemical, 13196), 50 μM, 24 hours prior to imaging; Arp2/3 inhibitor CK-666 (Millipore-Sigma, SML0006), 500 μM, 6 hours prior to imaging; actin polymerization inhibitor cytochalasin D (Cayman Chemical, 11330), 10 μM, 1 hour prior to imaging.

Following guidelines in IRB-approved protocol 02-060, primary myxofibrosarcoma cell lines (MXF8500, MXF2734, MXF8000, and MXF9100) were derived during 2004 to 2011 from fresh human primary myxofibrosarcoma tumor samples using collagenase digestion and established in cell culture. Array CGH was performed on all primary cell lines and compared to array CGH performed on the human tumor tissue from which they were derived so as to verify that the copy number alterations in the cell lines were representative of those found in the original tumor samples. The cell lines were last tested in 2014.
Adipose-derived stem cells (ASCs) were a generous gift from Dr. Jeffrey M. Gimble, and SGBS cells were a generous gift from Dr. Martin Wabitsch. Umbilical cord-derived human mesenchymal stem cells and human dermal fibroblast cells (KEL-FIB) were from ATCC. All cells were grown in a 50:50 mixture of Dulbecco’s modified Eagle’s medium (DMEM) high glucose and F12 medium (DMEM HG/F12) with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin and maintained in a 37°C incubator with 5% CO₂. Collagen I was from Sigma and Collagen II from Millipore. NSC23766 and INK128 were obtained from Cayman, EHop-016 from Millipore and Selleckchem, IPA3 from Tocris.

Drugs were reconstituted and stored following manufacturers’ recommendations and diluted in culture media to working concentrations. Specifically, 20 μM pan-MMPs inhibitor GM6001(Abcam, ab120845), 30 μM ROCK inhibitor Y27632 (Abcam, ab144494), 10 μM Cdc42 inhibitor ML141 (Calbiochem^®, 217708), 50 μM formin inhibitor SMIFH2 (Abcam, ab218296), 50 μM ARP2/3 inhibitor CK666 (Abcam, ab141231), 100 μM Rac1 inhibitor NSC23766, 20 μM Rac1 inhibitor EHT1864 (Cayman, 17258) were used. Fresh media mixed with specific drugs were prepared right before the experiment and replaced daily. In the drug assays, at least two independent experiments were performed with at least two replicates (technical duplicates) in each experiment.