MCF-7 and T47D cells stably expressing PAK1 WT (5×103 cells/well) were serum-deprived and allowed to grow for 7 days in 96-well plates. Each well contained 200 μl of DMEM (for MCF-7 clone) or RPMI (for T47D clone) were treated with either vehicle, 500 ng/ml of PRL, 1nM E2, 30 ng/ml HRG or 10 ng/ml EGF with or without 5 μM DIF-3(+1). In some experiments 5 μM IPA-3 (Tocris) was added. Cell proliferation was assessed by MTT cell proliferation assay (Life Technologies) according to manufacturer’s protocol.
Ipa 3
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
IPA-3 is a small molecule inhibitor that targets the p21-activated kinase (PAK) family of enzymes. PAK enzymes play a role in various cellular processes, including cytoskeletal reorganization, cell motility, and cell survival. IPA-3 functions by inhibiting the enzymatic activity of PAK proteins.
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Lab products found in correlation
β actin, by Merck Group (4 mentions)
Dasatinib, by Selleck Chemicals (4 mentions)
Nsc23766, by Bio-Techne (3 mentions)
Frax597, by Bio-Techne (3 mentions)
Mtt cell proliferation assay, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Bio-Techne (2 mentions)
Nsc23766, by Cayman Chemical (2 mentions)
Y 27632, by Merck Group (2 mentions)
Ab76293, by Abcam (2 mentions)
Ab223849, by Abcam (2 mentions)
Olive oil, by Merck Group (1 mentions)
Olive oil, by Bio-Techne (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Mk 801, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
Pluronic acid, by Thermo Fisher Scientific (1 mentions)
25 protocols using ipa 3
1
Modulating Breast Cancer Cell Proliferation
2
Hepatic Fibrosis Induction and Treatment
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Mice were given intraperitoneal (i.p.) injections of 2 μl per g body weight CCl4 (Sigma) at ratio of 1:3 by volume in olive oil (Sigma) or olive oil alone (control) twice weekly for 8 weeks.
For the VP (VP) experiments, fibrosis was induced by CCl4 injections for 6 weeks with VP treatment for the final 3 weeks. VP was dissolved in 10% DMSO in PBS to a concentration of 10 mg ml−1. Ten microlitre per g body weight (100 mg VP/kg bodyweight) was injected i.p. three times per week (on alternate days to CCl4 or olive oil). Control mice were injected with DMSO. This created four treatment groups: olive oil+DMSO (n=5); olive oil+VP (n=3); CCl4+DMSO (n=3); and CCl4+VP (n=4).
Treatment with IPA3 (Tocris Bioscience) or DMSO (Sigma) occurred during the final 4 weeks. IPA3 was dissolved in DMSO to a concentration of 4 μg μl−1; 1 μl per g body weight (4 mg IPA3/kg bodyweight) was injected i.p. three times per week (on alternate days to CCl4 or olive oil injections). Control mice were injected with DMSO. This created four treatment groups: olive oil+DMSO (n=5); olive oil+IPA3 (n=5); CCl4+DMSO (n=5); and CCl4+IPA3 (n=5).
For the VP (VP) experiments, fibrosis was induced by CCl4 injections for 6 weeks with VP treatment for the final 3 weeks. VP was dissolved in 10% DMSO in PBS to a concentration of 10 mg ml−1. Ten microlitre per g body weight (100 mg VP/kg bodyweight) was injected i.p. three times per week (on alternate days to CCl4 or olive oil). Control mice were injected with DMSO. This created four treatment groups: olive oil+DMSO (n=5); olive oil+VP (n=3); CCl4+DMSO (n=3); and CCl4+VP (n=4).
Treatment with IPA3 (Tocris Bioscience) or DMSO (Sigma) occurred during the final 4 weeks. IPA3 was dissolved in DMSO to a concentration of 4 μg μl−1; 1 μl per g body weight (4 mg IPA3/kg bodyweight) was injected i.p. three times per week (on alternate days to CCl4 or olive oil injections). Control mice were injected with DMSO. This created four treatment groups: olive oil+DMSO (n=5); olive oil+IPA3 (n=5); CCl4+DMSO (n=5); and CCl4+IPA3 (n=5).
3
Neuronal Culture Reagents and Protocols
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Trypsin, penicillin, streptomycin, heat-inactivated fetal bovine serum, horse serum, and soybean Trypsin inhibitor were obtained from Atlanta Biologicals (Norcross, GA, USA). Minimum essential medium (MEM), deoxyribonuclease (DNase), poly-L-lysine, poly-D-lysine hydrobromide, cytosine arabinoside, NMDA, protease inhibitor cocktail, MK-801, and ifenprodil were purchased from Sigma (St. Louis, MO, USA). Pluronic acid and Fluo-3 AM were purchased from Molecular Probes (Eugene, OR, USA). TCN-201, IPA-3, and NSC23766 were purchased from Tocris (Bristol, UK). Pierce ECL kits (Thermo Fisher Scientific, Rockford, IL, USA), Neurobasal, and B-27 supplements were purchased from Invitrogen Corporation (Carlsbad, CA, USA); the p-PAK1 antibody (Thr 212) from Santa Cruz Biotechnology (Dallas, TX, USA); and the PAK1 antibody anti-rabbit IgG HRP-linked antibody from Cell Signaling Technology (Danvers, MA, USA). Brevetoxin-2 (PbTx-2) was isolated and purified from Karinia breve cultures at the Center for Marine Sciences at the University of North Carolina (Wilmington, NC, USA). QNZ-46 was a gift from SF Traynelis, Department of Pharmacology, Emory University, Atlanta, GA. The GluN2D subtype of NMDA receptor knockout mice was obtained from Daniel T. Monaghan, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska.
4
Lipid-based Nanoparticle Synthesis
5
Inhibition of PAK1/2/3 Signaling Pathways
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IPA-3 (#3622) and PIR3.5 (#4212) were purchased from Tocris Bioscience and dissolved in sterile dimethylsulfoxide (DMSO) to make 50 mM stock solutions. Working solutions were prepared by 10 fold dilution of the stock solution in 50 mM Tris, pH 8.0, immediately before use. The cell density was adjusted to 3 × 105 cells/ml for all experiments involving IPA-3 and PIR3.5 treatment. FRAX597 (#6029) was purchased from Tocris Biosciences and dissolved in sterile DMSO to make 10 mM stock solution. Working solution was prepared by dilution in cell culture medium.
Dasatinib was obtained from Selleckchem (#S1021), 200 µM stock solution was made in sterile DMSO. The antibodies against PAK1/2 are specified in Table1 . Other antibodies were purchased from the following providers: JMJD6 (sc 28348) and GFP (sc-9996) from Santa Cruz, β-actin (A5441) from Sigma-Aldrich, PAK3 from Cell Signaling (#2609). Anti-RFP was from Santa Cruz (sc-390909) or from Chromotek (6G6).
Dasatinib was obtained from Selleckchem (#S1021), 200 µM stock solution was made in sterile DMSO. The antibodies against PAK1/2 are specified in Table
6
Kinase Inhibitor Assay Protocol
7
Nef-Associated PAK2 Activity Assay
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To assess Nef-associated PAK2 activity, standard IVKAs were performed as previously described (35 (link)). Nef.GFP was immunoprecipitated from transfected Jurkat TAg cells after lysis in KEB (137 mM NaCl, 50 mM Tris/HCl [pH 8], 2 mM EDTA, 0.5% Nonidet P-40, 1 mM Na3VO4, protease inhibitors). Upon incubation with GFP-Trap beads, samples were extensively washed with KEB and resuspended in 50 µl of KAB (50 mM HEPES [pH 8], 150 mM NaCl, 5 mM EDTA, 0.02% Triton X-100, 10 mM MgCl2) containing 10 µCi of [γ-32P]ATP (Hartmann Analytic) per reaction. After 10 min of incubation at room temperature, samples were repeatedly washed in KEB, mixed with 2× SDS sample buffer, and boiled. Bound proteins were separated by SDS-PAGE and subjected to autoradiography. For pharmacological inhibitor experiments, GFP- or Nef.GFP-transfected Jurkat T lymphocytes were incubated for 1 h with 10 µM PAK2 kinase inhibitor IPA3 (catalog no. 3622; Tocris) or its inactive pharmacological control PIR3.5 (catalog no. 4212; Tocris) and then subjected to IVKA.
8
Micropillar Perturbant Delivery Protocol
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Chemical perturbants were acquired from Sigma-Aldrich, except SMHIF2 (Merck), calpeptin (Cytoskeleton), IPA3 and CK666 (Tocris). Perturbants were used at the indicated concentrations. A microsyringe (Hamilton) was used to inject the perturbants into the Petri dish containing the micropillar substrates. Supplementary Table 1 lists the perturbants used, how they were applied (preincubated or added after nuclear envelope breakdown) and a short description of their action.
9
Inhibitor Effects on Cell Proliferation
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Cells were seeded in 24 well plates and exposed to inhibitors [IPA-3 (Tocris Biosciences, Cat. number 3622); FRAX597 and FRAX1036 (Genentech)] at desired concentration. Cells from each well were harvested in triplicates daily and counted by hemocytometer. The mean of cell counts were plotted over the course of five days.
10
Optimizing miPSC Culture Conditions
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One day prior to miPSCs seeding (day 1), CFL-condition 96-well plates were coated with a mixture of 15 µg fibronectin (Cat# 356008, Corning Inc., Corning, NY, USA), 24 µg laminin/entactin (Cat# 354259, Corning Inc., Corning, NY, USA), and 61 µg collagen type I (Cat# 354249, Corning Inc., Corning, NY, USA), all mixed in deionized ultra-filtered water and incubated at 4 °C overnight. The next day (day 0), the plates were washed twice with sterile phosphate-buffered saline (PBS) prior to miPSC seeding. The gelatin condition was coated with 0.1 % gelatin for 5 min at room temperature before miPSCs seeding. miPSCs were plated in modified GMEM without LIF at 7000 cells/cm2. The next day (day 1), media was replaced with unmodified GMEM (Cat# G5154, Sigma-Aldrich, St. Louis, MO, USA) and changed every day for 2 weeks, with or without the FAK inhibitor, PF-573228 at 1 µM in DMSO (Cat# 3239, Tocris Bioscience, Bristol, UK); the ILK inhibitor, Cpd22 at 0.4 µM in DMSO (Cat# 407331, MilliporeSigma, Burlington, MA, USA); the PAK-1 inhibitor, IPA-3 at 1 µM in DMSO (Cat# 3622, Tocris Bioscience, Bristol, United Kingdom); or 0.1 % DMSO as vehicle control.
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