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: Theranostics

Article Title: An exosomal-carried short periostin isoform induces cardiomyocyte proliferation

doi: 10.7150/thno.57243

Figure Lengend Snippet: CPC-derived Exo induce neonatal cardiomyocyte cycling via periostin-mediated FAK activation. A. Western analysis of focal adhesion kinase (FAK) phosphorylation in neonatal cardiomyocytes treated with naïve CPC-derived Exo (ExoCPC), Exo from CPC transfected with a siRNA against periostin (ExoCPC_SiPOSTN), or PBS (Ctrl). Quantitative analysis of FAK phosphorylation (fold-changes over Ctrl; n = 4; * p =0.0187 and p =0.0220). B. Western analysis of F-actin and G-actin expression. Quantitative analysis of F-actin/G-actin ratio (fold-changes over Ctrl; n = 6; * p =0.0419 and p =0.0172). C. Quantitative analysis of F-actin intensity in cardiac troponin I (cTnI)-positive cells (fold-change over Ctrl; n = 3; * p =0.0356 and p =0.0455). D. Western analysis of the effect of the FAK phosphorylation inhibitor, PF-573228, on FAK phosphorylation. E. Quantitative analysis of the effect of PF-573228 on EdU-positive cardiomyocytes (fold-changes; n = 4 to 5; ** p =0.0064 and p =0.0048). F. Quantitative analysis of the effect of PF-573228 on phosphorylated histone H3 (pH3)-positive cardiomyocytes nuclei (fold changes; n = 4 to 6; ** p =0.0033 and p =0.0068).

Article Snippet: FAK phosphorylation inhibitor, PF-573228 (10 mM; Sigma), was added to cells 30 min before Exo treatment.

Techniques: Derivative Assay, Activation Assay, Western Blot, Transfection, Expressing

Journal: Theranostics

Article Title: An exosomal-carried short periostin isoform induces cardiomyocyte proliferation

doi: 10.7150/thno.57243

Figure Lengend Snippet: CPC-derived Exo induce in vitro and in vivo cardiomyocyte cycling via periostin-mediated YAP nuclear translocation. A. Cultured neonatal rat cardiomyocytes immunostained for YAP (red) and cardiac-specific α-actinin (sarcomeric; green); nuclear staining with DAPI (blue). Scale bars: 200 µm. Quantitative analysis of YAP fluorescence intensity in the nuclear fraction of cardiomyocytes treated with naïve CPC-derived Exo (ExoCPC), Exo from CPC transfected with a siRNA against periostin (ExoCPC_SiPOSTN), or PBS (Ctrl; n = 5; * p =0.0316; ** p =0.0049). B. Western analysis of YAP and histone H3 in the nuclear fraction of cardiomyocytes and PF-573228 pre-treated cardiomyocytes. Quantitative analysis of YAP levels normalized for H3 in the nuclear fraction (fold-changes over Ctrl; n = 4 to 6; * p =0.0240; ** p =0.0052). C. Knocking down of YAP expression in the nuclear fraction using a siRNA against YAP (SiYAP; fold-changes over naive cells; n = 3; *** p =0.0002). D. Quantitative analysis of the effect of SiYAP on EdU-positive cardiomyocytes (fold-changes over the respective controls; n = 4 to 5; ** p =0.0064 and p =0.0048). E. Quantitative analysis of the effect of SiYAP on phosphorylated H3 (pH3)-positive cardiomyocytes (fold-changes over the respective controls; n = 4 to 6; ** p =0.0033 and p =0.0068). F. Western analysis of YAP and H3 levels in dispersed isolated cardiomyocytes at day 14 after IP injection of ExoCPC, ExoCPC_SiPOSTN or PBS. Quantitative analysis of YAP levels normalized for H3 (fold-changes over Ctrl; n = 5; * p =0.0300 and p =0.0258). G. Western analysis of YAP and H3 levels in dispersed isolated cardiomyocytes from adult rat hearts explanted 14 days after MI and intramyocardial injection of ExoCPC, ExoCPC_SiPOSTN, or PBS. Quantitative analysis of YAP levels normalized for H3 (fold- changes over Ctrl; n = 6).

Article Snippet: FAK phosphorylation inhibitor, PF-573228 (10 mM; Sigma), was added to cells 30 min before Exo treatment.

Techniques: Derivative Assay, In Vitro, In Vivo, Translocation Assay, Cell Culture, Staining, Fluorescence, Transfection, Western Blot, Expressing, Isolation, Injection