Cp 673451

The sources of antibodies were as follows: PDGFRΒ (polyclonal, catalog number sc-432), vinculin (monoclonal, sc-73614), cyclin D1 (monoclonal, sc-8396), and cyclin A (polyclonal, sc-751) from Santa Cruz Biotechnology; SMAD4 (monoclonal, #38454, raised against peptide corresponding to residues surrounding D165), YAP1 (polyclonal, #4912), C/EBPα (polyclonal, #2295), FABP4 (polyclonal, #2120), PPARγ (#2430, raised against peptide corresponding to residues surrounding D69), and p-RB (Ser780) (polyclonal, #9307, raised against phosphopeptide corresponding to residues surrounding S780) from Cell Signaling Technology; and anti-RB (polyclonal, 09–100, raised against linear protein corresponding to human RB-like protein 2 at and around the C-terminus) from EMD Millipore. miRNAs (nonspecific miRNA, miR-193b) and antimiRNAs (nonspecific antimiRNA, anti-miR-193b) were purchased from Ambion. Smartpool siRNAs for SMAD4, PDGFRΒ and YAP1 were purchased from Dharmacon. CP-673451 and ICG-001 were purchased from Selleck Chemicals.

To assess drug screening experiments, cells in the microfluidic device were exposed to reported in vivo plasma concentrations of different FDA approved anti-cancer drugs or a standard dose of 100 nM. 5-Fluoruracil (5-FU), Vincristine, Sorafenib and Taxol were obtained from the NIH-MPS Training Compound Collection at Evotec. Oxaliplatin, Pazopanib, Linifanib, Apatinib and CP-673451 were purchased from Selleck Chemicals. Vemurafenib, Vandetanib, Cabozantinib and Sorafenib were obtained from National Cancer Institute (NCI) plate sets. All compounds were dissolved in dimethyl sulfoxide (DMSO) and added in the medium with less than 0.01% DMSO. For hormone response in breast cancer cell lines, Estradiol (Sigma-Aldrich) was used at physiological plasma concentration. Estradiol was dissolved in 100% ethanol and added in the medium with less than 0.001% Ethanol. For a typical screening assay, after 5–8 days of cells cultured in the microdevice, media from the device is replaced by media containing the drug at the desired concentration, and delivered through the microfluidic channels using the hydrostatic pressure gradient.

CP-673451 (S1536, Selleckchem, Houston, TX, USA), a selective PDGFR-β inhibitor, was used to deplete pericytes, as previously described [20 (link)]. Briefly, rats received CP-673451 at a dosage of 40 mg/kg per day or vehicle (polyethylene glycol 400) for 7 consecutive days via gastric gavage.

MK-2206, LY294002, BAY 11-7082, and CP-673451 were purchased from Selleck Chemicals (Houston, TX, USA). 666-15 was obtained from Tocris Bioscience (Bristol, UK). Puromycin was purchased from Solarbio (Beijing, China). Mouse PDGFA factor from Novus Biologicals (Colorado, USA). pLXIN-mutAKT1 (AKT1E17K), pLXIN-myrAKT1 (myristoylated AKT1) expression plasmids, and the empty control pLXIN vector have been described previously^{14 (link)}. The CREB S133A cDNA was amplified using pCF-CREB M1 plasmid (#22969, Addgene, Watertown, MA, USA) as templates and subcloned into a pLXIN retroviral vector. pGL3-Basic and pRL-TK plasmid were from Promega (Madison, WI, USA). PTEN (#9559), PDGFRα (#3174), PDGFRβ (#4564), p-AKT (Ser-473) (#4060), AKT1 (#2967), p-AKT1 (Ser-473) (#9018), CREB (#9197), p-CREB (Ser133) (#9198), p-PDGFRα^Y849/PDGFRβ^Y857 (#3170), cleaved caspase-3 (#9664), AKT2 (#3063), AKT3 (#3788), p-IκBα (#2859), IκBα (#4814), FOXO1 (#2880), FOXO3a (#2497), LaminB1 (#13435), Ki-67 (#12202), GAPDH (#2118) and β-actin (#4970) antibodies were from Cell Signaling Technology (Danvers, MA, USA). All horseradish peroxidase (HRP)-labeled secondary antibodies were from Jackson Immuno Research (West Grove, PA, USA).

TAS‐115 [4‐[2‐fluoro‐4‐[[[(2‐phenylacetyl)amino]thioxomethyl]amino]‐phenoxy]‐7‐methoxy‐N‐methyl‐6‐quinolinecarboxamide] was provided by Taiho Pharmaceutical Co., Ltd. (Tsukuba, Japan). CP‐673451 (PDGFR inhibitor), TP‐0903 (AXL inhibitor), and quizartinib [Fms‐like tyrosine kinase 3 (FLT‐3) inhibitor] were purchased from Selleck Chemicals (Houston, TX, USA). According to the manufacturer’s instructions, TAS‐115, CP‐673451, TP‐0903, and quizartinib were prepared in dimethyl sulfoxide (DMSO; Sigma‐Aldrich), before their addition to the cell cultures for the in vitro experiments. TAS‐115 was diluted in water with 2‐hydroxypropyl‐β‐cyclodextrin (HPβCD) to the appropriate concentrations for the in vivo experiments, according to the manufacturer’s instructions. Antibodies against platelet‐derived growth factor receptor alpha (PDGFRα) (#3174), p‐PDGFRα (Tyr⁸⁴⁹; #3170), p‐AXL ( #5724), AXL ( #8661), FLT‐3 (#3462), p‐FLT‐3 (#3461), poly(ADP‐ribose) polymerase (PARP) (#9542), CD31 (PECAM‐1) (#77699), and β‐actin (#4970), and anti‐rabbit IgG horseradish peroxidase‐linked secondary antibody (#7074) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against AXL (ab32828) was purchased from Abcam (Cambridge, UK).

Defined compounds used for validation experiments were ordered de novo from various suppliers: SB-431542, PD-173074, U0126, Salinomycin, Nigericin, PD-161570, PD-166285, PD-166866, Idoxuridine and 5-Azacytidine (all from Sigma); Y-27632 2HCl, GSK429286A, Nintedanib, Pazopanib, Sorafenib, Vatalanib, Axitinib, Sunitinib, Erlotinib, Lapatinib, Gefitinib, Dasatinib, Crizotinib, Vandetanib, AEE788, Cediranib, AZD4547, CP673451, TSU-68 and KX2-391 (all from Selleck Chemicals); PP1, PP2, Rho kinase inhibitor V, SR-3677, GSK269962, SB-772077B (all from Tocris); Fasudil from Enamine; CAY10622 and PD-166326 from Cayman; BGJ398 from Axon Medchem; 5-Aza-2′-deoxycytidine from Chem-Impex. The reordered compounds were tested in extended dilution series ranging from 25 μM to 0.04 nM on TGFβ-treated NMuMG cells and from 25 μM to 1 nM on TGFβ-treated Py2T cells.