Auy922

Small molecule inhibitors were purchased from Tocris Biosciences (R&D Systems): PF-4708671, U 73122, GSK2334470, IPA3, SL0101-1 and XMD 8-92 or from Selleck Chemicals LLC (TX, USA): PF-562271, Enzastaurin (LY317615), AUY922 (NVP-AUY922), 17-AAG (Geldanamycin), PF-04929113 (SNX-5422), AZD6244 (Selumetinib), AT7867, CHIR-98014, LY2228820, BIX 02188, AS703026, PH-797804, SP600125, NU7441. All inhibitors were prepaed in DMSO at 100 mM. Cells were treated with inhibitors prepared in culture media where the final concentration of DMSO was 2% v/v. Vehicle control treatments consisted of culture media containing 2% v/v DMSO. Six days after treatment, cell survival was measured in comparison to vehicle controls using the CellTiter 96® Assay as per manufacturer instructions (MTS assay, Promega Corporation, WI, USA). Data were analyzed in GraphPad Prism® version 5.00 for Windows (GraphPad Software, CA, USA) to measure the log₁₀ of IC50 for each drug. For combination assays, drugs were added simultaneously. Heatmaps for sensitivities (-log₁₀ of IC50) were prepared using D-chip Analyzer software [49 (link)].

The local animal ethics committee gave approval for use of the mice. Female balb/c nude mice at 5 weeks of age (Animal Resources Centre, WA, Australia) were inoculated in the mammary fat-pads with exponentially growing MDA-MB-231 TNBC cells (5×10⁶ per fat pad) in 50 µL of 50:50 PBS:Matrigel™ (BD Biosciences, CA, USA). Treatments started when tumors were 50 ± 1 mm³ as calculated from caliper measurement of tumor's longest (a) and shortest (b) diameters: tumor volume (mm³) = a/2 × b². Docetaxel, doxorubicin and the Hsp90 inhibitor AUY922 were purchased from Selleck Chemicals and the Erk5 inhibitor XMD 8-92 from Tocris Biosciences. All drugs were diluted in 5% solution of D-glucose in PBS for intraperitoneal injection. Doses and schedule of treatments are detailed in Figure legends. Mice were monitored for change in weight and other toxicity indications and none were observed. Tumor growth was monitored by caliper measurements to calculate the change in tumor volume compared to day 0. Six tumors were excised on day 13 to perform ex vivo studies as below.

AT9283, AZD4547, AZD6244, BGJ398, crizotinib, pictilisib, ibrutinib, PD173074, ruxolitinib, TAE684, and gandotinib were all purchased from Selleck Chemicals, AUY922, dovitinib, everolimus, gefitinib, mubritinib, saracatinib, sunitinib, and ZSTK474 were from LC Laboratories, CH5424802 was from Active Biochem, SB525334 was from Tocris, and fedratinib was from Axon Medchem. The human gastric cancer cell line SNU-16 was obtained from ATCC and was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum according to the manufacturer’s instructions.

17‐Allyl‐amino‐demethoxy‐geldanamycin (17‐AAG) was obtained from the National Cancer Institute (Bethesda, MD, USA). AUY‐922 was purchased from Selleckchem (Houston, TX, USA). P53 siRNA (sc‐29435), PAK siRNA (sc‐29700), P190RHOGAP siRNA (sc‐44077), PDXP siRNA (sc‐61425), control siRNA (sc‐37007) and MDM2 antibody (sc‐965) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p53 (9282s), p‐myosin light‐chain 2, cofilin (3318), phospho‐cofilin (3311), PAK1 (2602), LIMK1 (3842) and phospho‐LIMK1 (3841) antibodies were obtained from Cell Signaling (Danvers, MA, USA). Β‐actin antibody (P8999) and CelyticM lysis reagent (C2978) were purchased from Sigma‐Aldrich (St Louis, MO, USA). Secondary mouse and rabbit antibodies were purchased from Licor (Lincoln, NE, USA). Oligofectamine (12252011), Pierce BCA protein assay and nitrocellulose membranes were obtained from Fisher Scientific (Pittsburgh, PA, USA). Ad‐p53‐GFP (1260) and ad‐GFP (1060) were obtained from Vector Biolabs (Malvern, PA USA).

IVIgG and rituximab (RTX) were from The University of Texas MD Anderson Cancer Center Pharmacy (Houston, TX), and IVIgG was dialyzed against phosphate-buffered saline (PBS) to remove L-proline. AUY922, bortezomib (BZB), and carfilzomib (CFZ) were from Selleck Chemicals (Houston, TX), recombinant human HSP70-1 protein was from StressMarq Biosciences (British Columbia, Canada), and bovine serum albumin (BSA) was from Sigma-Aldrich (St. Louis, MO).