Journal: Frontiers in Cell and Developmental Biology

Article Title: MFG-E8 Maintains Cellular Homeostasis by Suppressing Endoplasmic Reticulum Stress in Pancreatic Exocrine Acinar Cells

doi: 10.3389/fcell.2021.803876

Figure Lengend Snippet: rhMFG-E8 alleviates ER stress in pancreatic exocrine acinar cells through integrin αVβ3/5-FAK-STAT3. Pancreatic AR42J cells (5 × 10 5 /well) were treated with 100 nmol/L cerulein and 10 ng/ml LPS with or without 100 ng/ml MFG-E8 for 24 h. To determine whether the protective effect of MFG-E8 in ER stress is mediated through integrin αVβ3/5-FAK-STAT3, 5 ng/ml cilengitide, a specific integrin αVβ3/5 antagonist, 1 μmol/L-PF-00562271, a specific FAK antagonist, 30 μmol/L-APTSTAT3-9R, a specific STAT3 antagonist, was administered with 100 ng/ml MFG-E8, respectively. (A,B) Western blot analysis of the expression of GRP78, phospho-IRE1α, IRE1α, CHOP, caspase-9 and cleaved caspase-9 in the AR42J cells; (C–H) Western blot analysis of the expression of phospho-FAK, FAK, phospho-STAT3 and STAT3 in the AR42J cells; (I,J) Western blot analysis of the expression of GRP78, phospho-IRE1α, IRE1α, CHOP, caspase-9 and cleaved caspase-9 in the AR42J cells. n = 6/group, error bars indicate the SEM; ∗ p < 0.05 versus Sham group; # p < 0.05 versus Vehicle group. MFG-E8, milk fat globule EGF factor 8; GRP78, glucose-regulated protein 78; CHOP, C/EBP homologous protein; LPS, Lipopolysaccharide.

Article Snippet: The AR42J cells were treated with 100 nmol/L cerulein (C6660, Solarbio, United States) and 10 ng/ml Lipopolysaccharide (LPS) (L-8880, Solarbio, United States) with or without 20 or 100 ng/ml MFG-E8 (2805-MF, RD System, Inc. Minnesota, United States) for 24 h. In additional groups of AR42J cells, Cilengitide (5 ng/ml, SELLECK, Texas, United States), a specific αvβ3/5 integrin inhibitor or PF00562271 (S2672, SELLECK, Texas, United States), a specific FAK inhibitor or APTSTAT3-9R (S8197, SELLECK, Texas, United States), a specific STAT3 antagonist, were administered together with 100 ng/ml MFG-E8.

Techniques: Western Blot, 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: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p < 0.05 vs. the untreated samples)

Article Snippet: Total and phosphorylated FAK were detected using a polyclonal rabbit anti-human FAK diluted 1:3000 (v/v) in TBST, and a monoclonal rabbit anti-human phospho-Tyr397-FAK (D20B1) diluted 1:3000 (v/v) in TBST, respectively (Cell Signalling Technologies).

Techniques: Phospho-proteomics, Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Journal: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p < 0.05 vs. the untreated samples)

Article Snippet: Total and phosphorylated FAK were detected using a polyclonal rabbit anti-human FAK diluted 1:3000 (v/v) in TBST, and a monoclonal rabbit anti-human phospho-Tyr397-FAK (D20B1) diluted 1:3000 (v/v) in TBST, respectively (Cell Signalling Technologies).

Techniques: Phospho-proteomics, Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: Figure 5C

Article Snippet: Accession numbers and DOI are listed in the key resources table.Key resources tableREAGENT or RESOURCESOURCEIDENTIFIERAntibodiesGoat Polyclonal Anti-Mouse IgG (H+L) HRP ConjugatePromegaCat#: W4021; RRID: AB_43083Goat Polyclonal Anti-Rabbit IgG (H+L) HRP ConjugatePromegaCat#: W4011; RRID: AB_430833Rabbit Polyclonal Anti-phospho-FAK (Tyr397)Cell Signaling TechnologyCat.

Techniques: Control, Virus, Recombinant, Expressing, Protease Inhibitor, Lysis, Transfection, Electron Microscopy, Immunodepletion, Clone Assay, Protein Purification, Endotoxin Assay, Bradford Assay, Staining, Mass Spectrometry, Mutagenesis, Software, Isolation