Cells were seeded on 24-well plates coated with fibronectin (1 μg/cm2) or collagen type I (5 μg/cm2) and cultured for 24 hours in DMEM containing 1% FBS. Cells were washed with PBS and fixed with 96% ethanol for 10 minutes at room temperature followed by staining with 0.1% crystal violet for 20 minutes at room temperature. After washing three times with distilled water, the cells were lysed with 1% Triton X-100 for 5 minutes and lysates were transferred to a 96-well plate. The absorbance was read at 595 nm. For inhibition experiments, cells were incubated with U0126 (20 µM; Sigma-Aldrich) or PF573228 (10 µM; Sigma-Aldrich). Data were normalized considering proliferation of wild-type cells as 100% on collagen and fibronectin.
Pf573228
Manufactured by Merck Group
Sourced in United States, Germany
PF573228 is a small molecule compound that functions as a potent and selective inhibitor of the protein kinase FAK (Focal Adhesion Kinase). It is commonly used in laboratory research settings to study the role of FAK in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Cytochalasin d, by Merck Group (4 mentions)
Blebbistatin, by Merck Group (3 mentions)
Y 27632, by Merck Group (3 mentions)
Ly294002, by Merck Group (3 mentions)
Tgf β1, by Thermo Fisher Scientific (3 mentions)
Nocodazole, by Merck Group (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
U0126, by Merck Group (2 mentions)
Pf 228, by Merck Group (2 mentions)
U0126, by Cell Signaling Technology (2 mentions)
Verteporfin, by Merck Group (2 mentions)
Dimethyl sulfoxoide, by Merck Group (2 mentions)
Sb 3ct, by MedChemExpress (2 mentions)
Pf 562 271, by Merck Group (2 mentions)
P aminophenylmurcuric acetate apma, by Merck Group (2 mentions)
Wortmannin, by Merck Group (2 mentions)
Y 27632, by Cayman Chemical (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Stattic, by Selleck Chemicals (2 mentions)
Gefitinib, by Selleck Chemicals (2 mentions)
68 protocols using pf573228
1
Cell proliferation on ECM coatings
2
Cytokine-Triggered Integrin Signaling Pathway
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Two types of pro-inflammatory cytokines, TNFα-human and TNFα-mouse (10 ng/ml; Sigma) as well as IL1β-human and IL1β-mouse (1 ng/ml; Sigma), were used. A function-blocking integrin β1 antibody (clone P5D2; 10 µg/ml; Santa Cruz Biotechnology) was used to block integrin activities. MβCD (10 mM; Sigma) was used to extract cholesterol from the lipid rafts of the plasma membrane. PP2 (10 µM; Sigma) was used to block SFK activities, and PF573228 (1 µM; Sigma) was used to inhibit FAK activities. Pyk2 siRNA and non-specific control (NC) siRNA (Santa Cruz Biotechnology) were used to investigate the role of Pyk2 in SFK and FAK activities.
3
miR-6875-3p Regulation of BTG2 Expression
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The shRNA targeting the BTG2, miR-6875-3p mimic, inhibitor and the negative control were obtained from Ribobio (Guangzhou, China). Cells (5 × 105 cells/2 ml/well) were plated at 60% confluence in a six-well plate in DMEM without antibiotics. After 48 h, miR-6875-3p mimic, inhibitor or negative control oligonucleotide was transfected into cells with Lipofectamine 2000 at a final concentration of 50 nM according to the instructions of the manufacturer’s. After 4–6 h, the medium was replaced with fresh DMEM containing 10% FCS and the cells were cultured for further experiment. The transfection of shRNA was performed as described above. To inhibit FAK phosphorylation, dissociated cells were incubated with PF573228 (10 μM, Sigma-Aldrich, Saint Louis, MO, USA) for 30 min at 37 °C before Western blot analysis.
4
Hypoxia and FAK Inhibition Impact on BMSCs
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1×105 BMSCs or 7×105 M210B4 cells/well were cultured overnight in 12-well plates (BD Falcon, Bedford, MA) in their respective media. The cells were treated with fresh medium supplemented with 100 μM of cobalt chloride (CoCl2) (Sigma-Aldrich, St. Louis, MO) for 48 h, following which the medium was replaced with fresh medium without CoCl2 (CoCl2-BMSCs) and the cultures were incubated under normoxia (21% oxygen, which is the standard cell culture condition). In hypoxia treatments, the cells were either continuously cultured under 1% oxygen (hypoxic-BMSCs) or under normoxia after preincubation for 48 h under hypoxia (BMSCs-48 h-hypoxia). For the treatment of focal adhesion kinase (FAK) inhibitor PF573228 (PF228; Sigma-Aldrich), overnight-cultured BMSCs (1×105/well of 12-well plate) were pretreated with 300 nM PF228 for 2 h, followed by the addition of 100 μM of CoCl2 for 48 h. Both compounds were removed before seeding the HSCs.
The cell viability in all cases was >98% as determined by Trypan Blue dye exclusion method.
The cell viability in all cases was >98% as determined by Trypan Blue dye exclusion method.
5
Investigating Signaling Pathways in Cell Proliferation
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Antibodies specific for CD147 (sc-9754), α-tubulin (sc-8035), WAVE2 (sc-10392), donkey anti-goat IgG-FITC (sc-2024) and FAK Inhibitor 14 (Y-15) (sc-203950) were purchased from Santa Cruz (Dallas, Texas); phospho-FAK (Tyr397) (3283), phospho-Src (Tyr416) (2013), phospho-myosin light chain 2 (Thr18/Ser19) (3674), Src (2109), and myosin light chain 2 (3672) antibodies were purchased from Cell Signaling Technology (Boston, MA); DOCK8 (11622-1-AP) and STAT3 (60199-1-Ig) antibodies were obtained from Proteintech (Wuhan, China); phospho-STAT3 (Tyr705) (ab76135) antibody was obtained from Abcam (Cambridge, UK); phospho-Src (Tyr527) (orb14869) antibody was obtained from Biorbyt LLC (San Francisco, CA); and Alexa 594-conjugated goat anti-mouse IgG and Alexa Fluor 488 phalloidin were purchased from Invitrogen (Carlsbad, CA). The Src kinase inhibitor Src I-1, FAK inhibitor PF573,228 (Sigma, St. Louis, MO) and STAT3 inhibitor WP1066 (Merck Millipore, Darmstadt, GER) were also used. Src I-1 was used at 300 nM, PF573,228 at 300 nM, Y-15 at 10 μM and WP1066 at 5 μM. The Cell Proliferation Reagent WST-1 was purchased from Roche (Mannheim, GER).
6
Regulation of EMT by NID1 via ERK/MAPK
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To evaluate the function of ERK/MAPK signaling pathway in the EMT-promoting role of NID1, OVCAR-3-NID1-MC cells were treated with 50 μM U0126 (an effective MEK inhibitor, Cell Signaling Technology, Danvers, MA, USA) for 24h. These cells were lysed and subjected to Western blot analysis.
To examine the role of FAK in the activation of ERK/MAPK signaling pathway by NID1, OVCAR-3-NID1-MC cells were treated with 5 nM PF573228 (Sigma-Aldrich, St.Louis, Missouri, USA) for 24h, which effectively inhibited FAK phosphoryation on Tyr397. These cells were lysed and subjected to Western blot analysis.
To examine the role of FAK in the activation of ERK/MAPK signaling pathway by NID1, OVCAR-3-NID1-MC cells were treated with 5 nM PF573228 (Sigma-Aldrich, St.Louis, Missouri, USA) for 24h, which effectively inhibited FAK phosphoryation on Tyr397. These cells were lysed and subjected to Western blot analysis.
7
Hypoxia/Reoxygenation Injury in H9C2 Cells
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As described previously, H9C2 cardiomyocytes were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco Laboratories) supplemented with 10% fetal bovine serum (FBS) (Gibco Laboratories) and 1% penicillin/streptomycin in an atmosphere of 90% air and 10% CO2 at 37 °C (19 (link)). The experiment was performed with the control, H/R, low-dose (2.5 µM Art), medium-dose (5 µM Art), and high-dose (10 µM Art) groups. To explore the effect of the Art and FAK pathway inhibitor PF573228 on H/R injury, another experiment was performed with the control, H/R, H/R + PF573228 group, the H/R + PF573228 + Art (10 µM Art) group. Inhibitors that do not affect the morphology or viability of H9C2 cells were used. Hypoxia was established by culturing H9C2 cells for 4 hours at a constant temperature three-gas incubator with a mixture of 95% N2, 5% CO2, and 1% O2 at 37 °C. After that, the cells were reoxygenated in a normoxic chamber for 2 hours. A myocardial cell model (H9C2 cells) of hypoxia/reoxygenation (H/R) was established.
The FAK inhibitor PF573228 was obtained from Sigma-Aldrich (St. Louis, MO) and dissolved with dimethyl sulfoxide (DMSO) to a final concentration of <0.1% per assay.
The FAK inhibitor PF573228 was obtained from Sigma-Aldrich (St. Louis, MO) and dissolved with dimethyl sulfoxide (DMSO) to a final concentration of <0.1% per assay.
8
Investigating Cell Migration Signaling
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Antibodies include mouse monoclonal for HA-probe (F-7) (sc-7392), β-actin (C4) (sc-47778), and GFP (sc-101525) and rabbit polyclonal for FAK (sc-557) (Santa Cruz Biotechnology Inc, Dallas, TX, USA), CXCR4 rabbit polyclonal Ab (Ab-2074) (Abcam, Cambridge, MA, USA), and pY397-FAK rabbit monoclonal Ab (Invitrogen, 44625G, Carlsbad, CA, USA), and vimentin mouse monoclonal Ab (550513) (BD Pharmingen, San Jose, CA, USA). Anti-KLF8 rabbit polyclonal Ab was described previously [18 (link), 19 (link)]. Peroxidase substrate kit (DAB) (SK-4100) was purchased from Vector laboratories Inc. (Burlingame, CA, USA). The CXCR4 inhibitor AMD3100 (or octahydrochloride hydrate) (A5602) and the FAK inhibitor PF573228 (PZ0117) were purchased from Sigma (St. Louis, MO, USA). Recombinant human CXCL12 (300–28A) was purchased from Peprotech (Rocky Hill, CT, USA).
9
Regulation of Runx2 by FAK, ROCK, and YAP in hMSCs
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To study the role of FAK, ROCK, and YAP in the regulation of Runx2 as an osteogenic marker in hMSCs while exposed to high aspect ratio nanopillars, each of those factors was inhibited in separate dedicated experiments. The pre-culture, cell seeding, and the composition of the culture medium were kept identical to the procedures described in Section 2.2.1 during these experiments while the following inhibitors were added to the culture medium upon cell seeding and medium refreshing: 10 μM PF-573228 (FAK inhibitor, Sigma-Aldrich, Germany), 10 μM Y-27632 (ROCK inhibitor, Abcam, The Netherlands), and 10 μM Verteporfin (YAP inhibitor, Sigma-Aldrich, Germany). The short-term adaptation of the cells to the surfaces under each new condition as well as the expression of a major osteogenic marker (i.e., Runx2) were evaluated through immunocytochemical staining procedures that were identical to those applied in Section 2.2.2 .
10
Investigating Dasatinib and MMP Inhibitors
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Dasatinib was from LC labs (Woburn, MA), SB-3CT was purchased from MedChem Express (Monmouth Junction, NJ). The human IL-1β neutralizing antibody was from R&D bio-techne. PF-562,271, PF-573,228, and p-aminophenylmurcuric acetate (APMA), for MMP activation, was purchased from Sigma (St. Louis, MO). All drugs were dissolved in dimethyl sulfoxoide (Sigma).
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