Myeloid hematopoietic precursors were expanded from BM cells using granulocyte-monocyte-colony stimulating factor (GM-CSF), stem cell factor (SCF) and leukaemia inhibitory factor (LIF) for 2 to 3 days, following published conditions [8 (link), 30 (link)]. Then, myeloid precursors were seeded on two L8 cell culture chambers for the xCELLingence RTCA monitoring system (ACEA biosciences), at a density of 200000 cells per well. DC or B16-MDSC differentiation medium was added to myeloid precursors, and treatments were carried out simultaneously in duplicates. After 30 min, the indicated chemical inhibitors were added at concentrations reported to be cytotoxic to cancer cells. Control wells were treated with carrier solution (either water or DMSO). The following inhibitors were used: AKT inhibitor X (Calbiochem), tyrosine kinase inhibitor TX-1123 (Calbiochem), MEK inhibitor PD0325901 (SIGMA), ERK inhibitors SCH772984 and VTX-11e [31 (link)], broad PKC inhibitor GÖ 6983 (Santa Cruz Biotechnology), PKC inhibitor NPC-15437 dihydrochloride (Santa Cruz Biotechnology), selective LCK and FYN inhibitor PP2 (Santa Cruz Biotechnology), and the SRC and FYN inhibitor Saracatinib (MedChem Express). IC50s for each inhibitor were calculated using the RTCA data and analysis software, using duplicates for each drug treatment.
Saracatinib
Manufactured by MedChemExpress
Sourced in China, United States
Saracatinib is a small molecule inhibitor that targets Src family kinases. It is a white crystalline powder and is used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Mek inhibitor pd0325901, by Merck Group (1 mentions)
Mut express 2 fast mutagenesis kit, by Vazyme (1 mentions)
Ab108312, by Abcam (1 mentions)
Pai 1, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Hek293t, by Thermo Fisher Scientific (1 mentions)
Aspc 1, by Thermo Fisher Scientific (1 mentions)
Panc 1, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Selleck Chemicals (1 mentions)
Wnt3a, by Abcam (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Xav 939, by MedChemExpress (1 mentions)
Dasatinib, by MedChemExpress (1 mentions)
24 well plate, by Corning (1 mentions)
Transwell insert, by BD (1 mentions)
Crystal violet, by Sangon (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Hindiii, by Takara Bio (1 mentions)
9 protocols using saracatinib
1
Myeloid Precursor Expansion and Differentiation Assay
2
GNAQ Mutant and ANXA2 Phosphorylation Study
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The GNAQ WT (without Flag‐tag fusion) and GNAQ T96S (with Flag‐tag fusion) plasmids were previously established.16 The plasmids for GNAQ WT, GNAQ T96S, GNAQ T96S 1–359, GNAQ T96S 1–247, GNAQ T96S 1–181, GNAQ T96S 182–247, and GNAQ T96S 248–359 were subcloned into pCDH‐CMV‐MCS‐EF1‐copGFP. Mutation in ANXA2 at tyrosine‐24 into aspartic acid (Y24D, to mimic hyperphosphorylation) or into phenylalanine (Y24F, to mimic hypophosphorylation) was carried out using the Mut Express II Fast Mutagenesis Kit following the manufacturer's instructions (Vazyme Biotech). All plasmid constructs were verified by sequencing. The GNAQ T96S peptide (AMQAMIRAMDSLKIPYKYEHN) fused with transcriptional activator protein (TAT) was synthesized using the Fmoc chemistry synthesis protocol as previously described.17 The Src inhibitor saracatinib (# HY‐10234) was purchased from MedChemExpress.
3
Saracatinib and PAI-1 Antagonist Protocol
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Saracatinib was purchased from MedChem Express (Shanghai, China). Human recombinant PAI‐1 and CCL5 (PeproTech, Rocky Hill, NJ, USA) were dissolved in culture medium, and a bioavailable PAI‐1 antagonist, PAI‐039 (PeproTech), was dissolved in DMSO (Sigma‐Aldrich) and stored at −80°C as a 1 mg/mL stock solution. The antibodies used included the following: BCRP/ABCG2 (Abcam, ab108312), PAI‐1 (Abcam, ab187262), CCL5 (Abcam, ab9666) and Ki‐67 (Abcam, ab15580).
4
Cell Culture of Human Pancreatic Cancer Lines
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Human pancreatic cancer cell lines PANC-1 (TCHu 98), AsPC-1 (TCHu 8), SW1990 (TCHu201), and CFPAC-1 (TCHu112) were purchased from the cell center of the Institute of Biochemistry and Cell Biology or National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences (Shanghai, China). HEK293T and HeLa cells were stored in our lab. HEK293T, HeLa, PANC-1, and AsPC-1 cells were cultured in Dulbecco’s modified Eagle’s medium (12500096, Gibco, USA). All culture mediums were supplemented with 10% fetal bovine serum (G10270-106, Gibco, USA), 100 U/ml penicillin G, and 100 μg/ml streptomycin (P1400, Solarbio) at 37 °C in a humidified incubator containing 5% CO2. The medium was replaced every 2–3 days and the cell was subcultured and used for an experiment at 80–90% confluence. Wnt agonist Wnt3a was purchased from Abcam (ab153563). Wnt inhibitor XAV939 was purchased from MedChemExpress (HY-15147). BLK kinase inhibitor saracatinib and LCK kinase inhibitor PP2 were both purchased from MedChemExpress (HY-10234 and HY-13805). Cycloheximide (CHX) and MG132 were purchased from Selleck (S7418 and S2619).
5
Saracatinib treatment in mouse model
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Six to eight weeks old C57BL/6J-CD45.2, B6.SJL-PTPRCA-CD45.1, FVB/NJ and Postn−/− mice (Gender Male/Female) were bred and maintained in the animal facility at IISER Thiruvananthapuram and KU Leuven. During the experiments, mice were maintained in isolator cages at humidified constant temperature (22 ± 1°C), with a 12 h light–dark cycle. The mice were fed with autoclaved water and irradiated food, ad libitum. At IISER TVM, the animals were maintained as per guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India. All animal experiments were approved by the Institutional Animal Ethics Committees for the respective animal facilities.
Animals were treated with vehicle or Saracatinib via oral gavage. Three doses of 25 mg/kg Saracatinib (Cat#HY-10234; MedChemExpress LLC, NJ, USA) were given on alternate days; vehicle contained 5% DMSO, 50% PEG300 and 45% H2O. To perform CFU-C assays and flow cytometry based analysis of donor-derived chimerism and HSPC analysis peripheral blood (PB) was collected by tail clipping method.
Animals were treated with vehicle or Saracatinib via oral gavage. Three doses of 25 mg/kg Saracatinib (Cat#HY-10234; MedChemExpress LLC, NJ, USA) were given on alternate days; vehicle contained 5% DMSO, 50% PEG300 and 45% H2O. To perform CFU-C assays and flow cytometry based analysis of donor-derived chimerism and HSPC analysis peripheral blood (PB) was collected by tail clipping method.
6
Transwell Migration Assay with Inhibitors
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Transwell experiments were performed as described previously [66 (link)]. PLC/PRF/5 or SK-Hep-1 cells under indicated treatments were deprived of serum for 24 h before analyses. A total of 1 × 105 cells were seeded to the upper chamber in transwell inserts (BD Biosciences) with serum-free medium, and medium containing 10% FBS was added into the lower chamber of 24-well plate (Corning). After additional 48 h, migrating cells adhered to the lower surface of filter were washed with PBS, fixed with 20% methanol for 20 min and stained with 0.1% crystal violet (Sangon Biotech). For SRC inhibitors treatment, cells were treated with PP2 (10 mM for 24 h, MedChemExpress) or Dasatinib (0.1 mM for 24 h, MedChemExpress) or Saracatinib (1 mM for 6 h, MedChemExpress) before seeding in chambers. The numbers of migrating cell per well were counted under a light microscope in eight predetermined fields. Data are presented as means ± SD of values from these eight fields.
7
Molecular Cloning and Transfection Workflow
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Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), Lipofectamine 3000, Lipofectamine RNAi Max, and puromycin were purchased from Thermo Fisher (Carlsbad, CA, USA). The restriction enzymes BamHI, EcoRI, HindIII, KpnI, and SacI were purchased from TaKaRa (Shiga, Japan). BFA, Saracatinib, and PP1 were obtained from MedChem Express Co., Ltd. (Shanghai, China).
8
Src Kinase Inhibition Assay
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The Src kinase activity was measured with or without puerarin (dissolved in DMSO) by using ADP-Glo™ kinase luminescent assay combined with Src Kinase Enzyme System according to the manufacture's protocol. Saracatinib (MedChemExpress, hy-10234，USA) was used as a positive Src kinase inhibitor. Relative Luminescence Unit (RLU) was recorded with a microplate reader (BioTek Synergy H1, USA) upon incubation with Kinase Detection Reagent. The IC50 of Src kinase activity was calculated by fitting the experimental data to Prism 8.0.
9
Investigating Inflammatory Markers and Signaling Pathways
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The antibodies used for immunoblotting included: mouse monoclonal antibody against GAPDH (RM2002; Beijing Ray, Beijing, China); rabbit antibodies against Fg (ab189490; Abcam, Cambridge, MA), pan-Akt (4691; Cell Signaling Technology, Danvers, MA), p-Akt (Ser473) (4060; Cell Signaling Technology), and goat anti-mouse (R3001, Beijing Ray) or goat anti-rabbit (R3002, Beijing Ray) horseradish peroxidase–conjugated secondary antibody.
The antibodies used for immunohistochemical staining included: CD11b (ab133357; Abcam), S100A9 (73425; Cell Signaling Technology), MPO (ab9535; Abcam), F4/80 (ab111101; Abcam), and CD31 (3528; Cell Signaling Technology).
Other reagents included DSS (36,000–50,000 kD; MP Biomedicals, Santa Ana, CA), Evans blue (E2129; Sigma-Aldrich, St. Louis, MO), Fg (F3879; Sigma-Aldrich), GPRP acetate, LY294002, taselisib, capivasertib, MK 2206, defactinib, Y15, saracatinib, WH-4-023, L-NIO, L-NMMA, ML-7, MLCK inhibitor, docetaxel, Jasplakinolide, and Cytochalasin D (MedChemExpress, Monmouth Junction, NJ).
The antibodies used for immunohistochemical staining included: CD11b (ab133357; Abcam), S100A9 (73425; Cell Signaling Technology), MPO (ab9535; Abcam), F4/80 (ab111101; Abcam), and CD31 (3528; Cell Signaling Technology).
Other reagents included DSS (36,000–50,000 kD; MP Biomedicals, Santa Ana, CA), Evans blue (E2129; Sigma-Aldrich, St. Louis, MO), Fg (F3879; Sigma-Aldrich), GPRP acetate, LY294002, taselisib, capivasertib, MK 2206, defactinib, Y15, saracatinib, WH-4-023, L-NIO, L-NMMA, ML-7, MLCK inhibitor, docetaxel, Jasplakinolide, and Cytochalasin D (MedChemExpress, Monmouth Junction, NJ).
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