fak-specific inhibitor pf573228  (Millipore)

Journal: The Journal of Biological Chemistry

Article Title: The Matricellular Protein Cyr61 Is a Key Mediator of Platelet-derived Growth Factor-induced Cell Migration *

doi: 10.1074/jbc.M114.623074

Figure Lengend Snippet: The PDGF-BB-Cyr61 pathway, but not the p38 or Akt pathways, mediated PDGF-BB-induced FAK activation, which leads to SMC migration. A, time course of PDGF-BB-induced FAK activation. Quiescent SMCs were stimulated with PDGF-BB (10 ng/ml) for various times. Cell lysates were subjected to Western blot analysis using antibodies against phosphorylated FAK at specific sites. β-actin was used as the loading control. B, Western blotting data show that knockdown of Cyr61 expression with specific Cyr61 siRNA blocked PDGF-BB-induced FAK activation. Non, non-silencing siRNA. C, pretreatment with the specific FAK inhibitor PF573228 (PF) dose-dependently blocked PDGF-BB-induced SMC migration. **, p < 0.01 versus control; ##, p < 0.01 versus the PDGF alone group. D, knockdown of FAK expression with the specific FAK siRNA blocked PDGF-BB-induced SMC migration. FAK siRNA knockdown efficiency is shown (inset). **, p < 0.01 versus control; ##, p < 0.01 versus PDGF/non-silencing siRNA group. E, effects of knockdown of Cyr61, FAK, or both Cyr61 and FAK on PDGF-induced SMC migration. **, p < 0.01 versus control; ##, p < 0.01 versus PDGF/non-silencing siRNA group. F, pretreatment with U0126 (10 μm) or SP600125 (SP) (10 μm) blocked PDGF-BB-induced FAK activation (Western blot analysis). DMSO, dimethyl sulfoxide. G, neither the specific PI3K inhibitors wortmannin (0.75 μm) or LY294002 (LY) (50 μm) nor the specific p38 inhibitor SB203580 (SB) (10 μm) had any significant effect on PDGF-BB-induced FAK activation.

Article Snippet: The FAK-specific inhibitor PF573228 was from TOCRIS Bioscience (Bristol, UK).

Techniques: Activation Assay, Migration, Western Blot, Control, Knockdown, Expressing

Journal: Scientific Reports

Article Title: A machine learning pipeline revealing heterogeneous responses to drug perturbations on vascular smooth muscle cell spheroid morphology and formation

doi: 10.1038/s41598-021-02683-4

Figure Lengend Snippet: Inhibition of FAK phosphorylation differentially affects VSMC spheroid formation and morphology. Human VSMCs treated with FAK inhibitor (PF573228) or DMSO (vehicle control) in high-glucose DMEM containing 10% FBS were incubated for 24 h to generate human VSMC spheroids. Total cell lysates were immunoblotted ( A ) for phosphorylated FAK at Tyr397 (pFAK) and GAPDH. The bar graph displays the pFAK levels normalized to the DMSO control ( B ). ( C ) Cultures were imaged using an upright microscope. n = 3 ( A – B ) and n = 12 ( C ) biological replicates were used. ***p < 0.001.

Article Snippet: For the FAK, Rac, Rho, and Cdc42 pharmacologic inhibitor experiments, cells in suspension culture were treated with 10 μM FAK-specific inhibitor PF573228 (Sigma), 5–20 μM Rac-specific inhibitor EHT1864 (Tocris), 5–20 μM RhoA-specific inhibitor Rhosin (Tocris), or 5–10 μM Cdc42-specific inhibitor ML 141 (Tocris) reconstituted in dimethyl sulfoxide (DMSO) for selected times up to 24 h.

Techniques: Inhibition, Incubation, Microscopy

Journal: Scientific Reports

Article Title: A machine learning pipeline revealing heterogeneous responses to drug perturbations on vascular smooth muscle cell spheroid morphology and formation

doi: 10.1038/s41598-021-02683-4

Figure Lengend Snippet: Initial morphological clustering analysis identifying distinct clusters in response to inhibitors of FAK, Rac, Rho, and Cdc42. ( A ) Four morphological features extracted from human VSMC spheroid images are displayed on a UMAP plot. The different colored markers are used to represent the various drug treatments: FAK (PF573228), Rac (EHT1864), Rho (Rhosin), and Cdc42 (ML141). ( B ) The silhouette values and the number of clusters on the training set were evaluated by varying the number of nearest samples. ( C ) Silhouette plots for the clustering results on the testing set. ( D ) Four morphological clusters plotted on the UMAP plot were observed based on the “roundness of the spheroid”. The different colored markers are used to represent the various morphological clusters with the drug treatments. The most representative images were overlayed on each cluster (2 images per cluster) in the UMAP plot. Clusters #1 and #3 showed spheroids with circular morphologies, and the spheroids in Clusters #2 and #4 showed noncircular and dispersed/disrupted morphologies. Only the samples in the testing set were used. ( E ) Average proportionality plot of the distribution of VSMC spheroids in each morphological cluster with the drug treatments from the repeated random splitting of training and testing sets. Only testing sets were used.

Article Snippet: For the FAK, Rac, Rho, and Cdc42 pharmacologic inhibitor experiments, cells in suspension culture were treated with 10 μM FAK-specific inhibitor PF573228 (Sigma), 5–20 μM Rac-specific inhibitor EHT1864 (Tocris), 5–20 μM RhoA-specific inhibitor Rhosin (Tocris), or 5–10 μM Cdc42-specific inhibitor ML 141 (Tocris) reconstituted in dimethyl sulfoxide (DMSO) for selected times up to 24 h.

Techniques:

Journal: bioRxiv

Article Title: Machine learning reveals heterogeneous responses to FAK, Rac, Rho, and Cdc42 inhibition on vascular smooth muscle cell spheroid formation and morphology

doi: 10.1101/2020.01.30.927616

Figure Lengend Snippet: Human VSMC spheroids were made using a hanging drop technique with 2000 cells per droplet in the presence of DMSO (vehicle control), (A-C) PF573228 (PF, FAK inhibitor), (D) EHT1864 (Rac inhibitor), (E) Rhosin (Rho inhibitor), or (F) ML141 (Cdc42 inhibitor) in high glucose DMEM containing 10% serum. Total cell lysates were immunoblotted (A) for phosphorylated FAK at Tyr397 (pFAK) and GAPDH. The bar graph shows pFAK levels normalized to DMSO control (B). Cultures were imaged after 24 hours of incubation using an upright microscope (C and F). n =4 (A), n =7 (C), n =4 (D), n =4 (E), and n =4 (F) biological replicates were used. p ***< 0 . 001 .

Article Snippet: For FAK, Rac, Rho, and Cdc42 pharmacologic inhibitor experiments, cells in suspension culture were treated with 10 μM FAK specific inhibitor PF573228 (Sigma)[ ], 5-20 μM Rac specific inhibitor EHT1864 (Tocris) [ ], 5-20 μM Rho specific inhibitor Rhosin (Tocris)[ ], or 5-10 μM Cdc42 specific inhibitor ML 141 (Tocris)[ ] reconstituted in Dimethyl sulfoxide (DMSO) for selected times up to 24 hours.

Techniques: Incubation, Microscopy

Journal: bioRxiv

Article Title: Machine learning reveals heterogeneous responses to FAK, Rac, Rho, and Cdc42 inhibition on vascular smooth muscle cell spheroid formation and morphology

doi: 10.1101/2020.01.30.927616

Figure Lengend Snippet: Mouse VSMC spheroids were made in the presence of either DMSO (vehicle control) or 10 µM PF573228 (PF) in high glucose DMEM containing 10% serum. Total cell lysates were immunoblotted (A) for phosphorylated FAK at Tyr397 (pFAK) and GAPDH. The bar graph displays pFAK levels normalized to DMSO control (B). (C) Cultures were imaged after 24 hours of incubation using an upright microscope. n =3 (A) and n =12 (C) biological replicates were used. * p < 0 . 05 .

Article Snippet: For FAK, Rac, Rho, and Cdc42 pharmacologic inhibitor experiments, cells in suspension culture were treated with 10 μM FAK specific inhibitor PF573228 (Sigma)[ ], 5-20 μM Rac specific inhibitor EHT1864 (Tocris) [ ], 5-20 μM Rho specific inhibitor Rhosin (Tocris)[ ], or 5-10 μM Cdc42 specific inhibitor ML 141 (Tocris)[ ] reconstituted in Dimethyl sulfoxide (DMSO) for selected times up to 24 hours.

Techniques: Incubation, Microscopy

Journal: bioRxiv

Article Title: Machine learning reveals heterogeneous responses to FAK, Rac, Rho, and Cdc42 inhibition on vascular smooth muscle cell spheroid formation and morphology

doi: 10.1101/2020.01.30.927616

Figure Lengend Snippet: Human VSMCs treated with (A) 10μM PF573228, (B) 5-20 μM EHT1864, (C) 5-20 μM Rhosin, (D) 5-10 μM ML141, or DMSO were used to generate human VSMC spheroids. Total cell lysates were immunoblotted for N-cadherin and GAPDH. n =5 biological replicates were used. p **< 0 . 05 or p ***< 0 . 001 .

Article Snippet: For FAK, Rac, Rho, and Cdc42 pharmacologic inhibitor experiments, cells in suspension culture were treated with 10 μM FAK specific inhibitor PF573228 (Sigma)[ ], 5-20 μM Rac specific inhibitor EHT1864 (Tocris) [ ], 5-20 μM Rho specific inhibitor Rhosin (Tocris)[ ], or 5-10 μM Cdc42 specific inhibitor ML 141 (Tocris)[ ] reconstituted in Dimethyl sulfoxide (DMSO) for selected times up to 24 hours.

Techniques:

Journal: bioRxiv

Article Title: Machine learning reveals heterogeneous responses to FAK, Rac, Rho, and Cdc42 inhibition on vascular smooth muscle cell spheroid formation and morphology

doi: 10.1101/2020.01.30.927616

Figure Lengend Snippet: Human VSMCs treated with inhibitors of FAK (PF573228), Rac (EHT1864), Rho (Rhosin), or Cdc42 (ML141), and DMSO (vehicle control) were used to generate human VSMC spheroids. Spheroids were imaged after 24 hours of incubation using an upright microscope.

Article Snippet: For FAK, Rac, Rho, and Cdc42 pharmacologic inhibitor experiments, cells in suspension culture were treated with 10 μM FAK specific inhibitor PF573228 (Sigma)[ ], 5-20 μM Rac specific inhibitor EHT1864 (Tocris) [ ], 5-20 μM Rho specific inhibitor Rhosin (Tocris)[ ], or 5-10 μM Cdc42 specific inhibitor ML 141 (Tocris)[ ] reconstituted in Dimethyl sulfoxide (DMSO) for selected times up to 24 hours.

Techniques: Incubation, Microscopy

Journal: bioRxiv

Article Title: Machine learning reveals heterogeneous responses to FAK, Rac, Rho, and Cdc42 inhibition on vascular smooth muscle cell spheroid formation and morphology

doi: 10.1101/2020.01.30.927616

Figure Lengend Snippet: (A) Four morphological features extracted from human VSMC spheroid images were plotted as UMAP plot. Different colored ‘*’s are used to represent the various drug treatments; FAK (PF573228), Rac (EHT1864), Rho (Rhosin), and Cdc42 (ML141). (B) The number of nearest samples between 2 and 200 were evaluated and the silhouette values were plotted. A cluster number of four was selected. (C) Silhouette plots for the clustering results. (D) Four morphological clusters plotted on the Umap were observed based on the “roundness of the spheroid”. Different colored ‘*’s are used to represent the various morphological clusters with treatments. The most representative images were inserted into each cluster (2 images per cluster) in the UMAP. Clusters 1 and 3 showed spheroids with circular morphology and the spheroids in clusters 2 and 4 showed disrupted/dispersed or irregular morphologies. (E) A proportionality plot was graphed to identify the distribution of spheroids in each morphological cluster with treatments.

Article Snippet: For FAK, Rac, Rho, and Cdc42 pharmacologic inhibitor experiments, cells in suspension culture were treated with 10 μM FAK specific inhibitor PF573228 (Sigma)[ ], 5-20 μM Rac specific inhibitor EHT1864 (Tocris) [ ], 5-20 μM Rho specific inhibitor Rhosin (Tocris)[ ], or 5-10 μM Cdc42 specific inhibitor ML 141 (Tocris)[ ] reconstituted in Dimethyl sulfoxide (DMSO) for selected times up to 24 hours.

Techniques:

Journal: Scientific Reports

Article Title: Hydrogen Sulfide Recruits Macrophage Migration by Integrin β1-Src-FAK/Pyk2-Rac Pathway in Myocardial Infarction

doi: 10.1038/srep22363

Figure Lengend Snippet: ( a ) RAW264.7 cells were incubated for 1 h in the presence or absence of PF431396 (5 μM), PF573228 (5 μM), PP2 (10 μM) or NSC23766 (25 μM) respectively, followed by incubation with additional 100 μM NaHS for 6 h. The migratory ability of RAW264.7 cells was determined by transwell assay. Scale bar, 50 μm. ( b ) The numbers of the migrated cells per field were shown as the mean ± SEM from three independent experiments. # P < 0.05 versus Control, **P < 0.01 versus NaHS treatment. ( c ) RAW264.7 cells were incubated with PF431396 (5 μM), PF573228 (5 μM) or PP2 (10 μM) respectively for 1 h before 100 μM NaHS treatment. The expressions of indicated proteins were assessed by western blot (I, total cell lysates; II, membrane extracts; p, phosphorylated; t, total).

Article Snippet: NaHS was administered instead of H 2 S. NaHS and the Pyk2 inhibitor (PF431396) were obtained from Sigma-Aldrich; The FAK specific inhibitor (PF573228), the Src inhibitor (PP2), and the Rac1 inhibitor (NSC23766) were purchased from Selleck Chemicals.

Techniques: Incubation, Transwell Assay, Control, Western Blot, Membrane