Nsc87877

The assays were performed as previously described [57 (link)]. Briefly, polarized T84 epithelial cells that were pretreated with or without the SHP inhibitor NSC87877 (20 μM, EMD Millipore) for 1 h were incubated apically with GC (MOI = 10) at 37°C for 6 h. The basolateral media were collected and cultured, and the resulting colonies were counted as transmigrated bacteria.

Recombinant human PDGF‐BB and EGF were purchased from Peprotech. 4‐OHT was purchased from Sigma. IIB‐08 and 11a‐1 were provided by Z.Y. Zhang (Purdue University). GS‐493 was provided by J. Rademann (Freie Universität Berlin) or was purchased from Bioduro. NSC‐87877 was purchased from Millipore. SHP099 was purchased from Alputon Inc.

Preadipocyte cell line 3T3-L1 was obtained from the ATCC Company (ATCC-CL-173, Manassas, USA), and cells were cultured in DMEM containing 10% (v/v) heat-inactivated bovine calf serum (1438645, Gibco, Life, Waltham, MA, USA) at 37 °C in a humidified incubator with 5% CO₂. The growth medium was changed every 2 days. For the differentiation induction, confluent 3T3-L1 cells (defined as day 0) were treated with 0.5 mM IBMX (Sigma, St Louis, MO, USA, I-7018), 1 μM Dex (Sigma, D-4902), 10 μg/ml INS (Sigma, I-5500) and 10% fetal bovine serum (FBS; SV3008702, Hyclone, Thermo Scientific, Waltham, MA, USA) for 2 days. The medium was replaced with DMEM containing 10 μg/ml INS and 10% FBS. After 2 days, cells were cultured in growth medium again for 4 days. For treatment with Shp2 activity inhibitors, cells were incubated with 10 μM PHPS1 (Sigma, P0039) or NSC87877 (Millipore, Billerica, MA, USA, 565851) in growth medium for the indicated time.

Breast cancer cell lines MCF7 and Bcap37 were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Both cell lines were ER positive. Cells were grown in normal DMEM with 10% FBS, 100 unit/mL penicillin and 100 µg/mL streptomycin. Prior to all experiments, cells were pretreated for at least 1 week with an estrogen-free medium (phenol red-free DMEM with 10% dextran-coated charcoal serum, 100 units/mL penicillin, 100 µg/mL streptomycin, 0.5 mM sodium pyruvate, and 2 mM l-glutamine) to prevent the effects of serum-derived estrogenic compounds. Cell transfection was carried out using Lipofectamine 2000 (Cat. 11668, Invitrogen) according to the manufacturer's instructions.
E2 (E8875, Sigma) was prepared as a 10 mM solution with ethanol and stored at −80°C. Tamoxifen (T5648, Sigma) or toremifene (T7204, Sigma) was dissolved in 10 mM DMSO. Phps1 (P0039, Sigma,) was dissolved in PBS solution. NSC87877 (565851, Millipore) and phps4 (a gift from Dr. Yuehai Ke (Department of Medicine, Zhejiang University) were dissolved in DMSO. Plasmids expressing siRNAs of Shp2 Plvth-H1, Plvth-H2, and Plvth-H3, as well as a control plasmid Plvth were also prepared as previously described [53] (link). Unless otherwise indicated, all other chemicals were obtained from Sigma/Fisher.

Phenyl vinyl sulfonate and phenyl vinyl sulfone (PVS) were purchased from Enamine Ltd (Monmouth, NJ). JTT-551 (PTP1B and TCPTP inhibitor; IC50 = 0.2 and 2 µM, respectively), NSC-87877 (SHP-1 and SHP-2 inhibitor; IC50 = 0.36 and 0.32 µM, respectively), and CX08005 (PTP1B and TCPTP inhibitor; IC50 = 0.78 and 0.48 µM, respectively) were obtained from Millipore Sigma (Darmstadt, Germany). β-actin mouse monoclonal antibody (clone AC-74, catalog number A5316) was obtained from Sigma-Aldrich (St. Louis, MO). STAT1 (rabbit monoclonal, catalog number 9175), phospho-STAT1 (tyrosine 701, rabbit monoclonal, catalog number 9167), phospho-STAT3 (tyrosine 705, rabbit monoclonal, catalog number 9131), Caspase-3 (rabbit monoclonal, catalog number 14220), anti-biotin HRP linked (goat, catalog number 7075), anti-rabbit (polyclonal) and anti-mouse (polyclonal) antibodies and cytokines were purchased from Cell Signaling Technology (Danvers, MA). Sodium vanadate was dissolved in water, sterile filtered and stored at 4 °C until use (Hill III & Rice, 2018 (link)).