Journal: Toxicological Sciences

Article Title: Arsenic Stimulates Myoblast Mitochondrial Epidermal Growth Factor Receptor to Impair Myogenesis

doi: 10.1093/toxsci/kfaa031

Figure Lengend Snippet: Arsenic effects on EGFR and mitochondrial regulation of nuclear cyclinD1. A, Representative images of cyclin D1 immunofluorescence in undifferentiated RC replated in arsenic-medium. AG-1478 was added simultaneously with arsenic in the differentiation protocol. B–D, Quantitative comparison of nuclear cyclin D1 levels in replated RC. Groups of cells in (C) and (D) were treated with SS-31 or XJB-5-131 after replating and all groups in (B–D) were fixed and analyzed after 3 days in arsenic-free culture. Representative images of cells from (C) and (D) are provided in Supplementary Figure 2. All experiments were repeated 3 times and the data are presented as mean ± SEM of relative fluorescence per cell captured in 5 separate images. Group comparisons were made using ANOVA followed by Tukey’s post hoc test for significance (*p < .05, **p < .01 relative to control, ^p < .05, ^^p < .01 relative to arsenic).

Article Snippet: Cells were then blocked with 5% donkey serum (Millipore, S30-100KC) diluted in PPB for 60 min, and then washed again 3 times with PBB before adding primary antibodies for 60 min. Primary antibodies recognized: Cyclin D1, (1:100, R&D Systems AF4196), pY 845 EGFR (1:150, Cell Signaling Technology 2231), and MTCO2 (1:200, ThermoFisher Scientific 12C4F12).

Techniques: Immunofluorescence, Comparison, Fluorescence

Journal: Toxicological Sciences

Article Title: Arsenic Stimulates Myoblast Mitochondrial Epidermal Growth Factor Receptor to Impair Myogenesis

doi: 10.1093/toxsci/kfaa031

Figure Lengend Snippet: Machine-learning modeling of arsenic adverse outcomes. Machine-learning models were built using different classifiers of arsenic signaling data in order to predict whether an individual cell belonged to the control conditions, an arsenic group, and/or an intervention group. The input data consisted of the pY845EGFR, MTCO2, or cyclin D1 protein expression levels and pY845EGFR/MTOC colocalization. The capacity of these single-cell markers to discriminate the experimental conditions was comparatively tested by several classifier models using cross-validation of the training set according to the various metrics. Table I: The training set (approximately two-third of the experimental data) was used to construct features attuned to the molecular fingerprint of the noncanonical EGFR pathway and combine them into a generalizable classifier model learned on the empirical dataset, and applied to the test dataset (ie, the remaining one-third of the experimental data) to evaluate the model’s predictive accuracy. Table II: To account for the potential statistical limitations caused by the relatively small training dataset and possible misrepresentation of the overall cell population heterogeneity, a bootstrapping approach of the original empirical data was utilized to generate a larger sample size. The increased accuracy of estimating errors in the classified data is described by the confusion matrices (A and B). Table III: The condition-labeled datasets and scores of the classification attributes according to their correlation with the class using Gini coefficient, information gain, ANOVA, and χ2.

Article Snippet: Cells were then blocked with 5% donkey serum (Millipore, S30-100KC) diluted in PPB for 60 min, and then washed again 3 times with PBB before adding primary antibodies for 60 min. Primary antibodies recognized: Cyclin D1, (1:100, R&D Systems AF4196), pY 845 EGFR (1:150, Cell Signaling Technology 2231), and MTCO2 (1:200, ThermoFisher Scientific 12C4F12).

Techniques: Expressing, Construct, Labeling

Journal: Carcinogenesis

Article Title: Wntless (GPR177) expression correlates with poor prognosis in B-cell precursor acute lymphoblastic leukemia via Wnt signaling.

doi: 10.1093/carcin/bgu166

Figure Lengend Snippet: Fig. 4. Wls expression is critical for Wnt-induced consequent downstream genes’ activation. (A) Ablated Wls protein expression in B-cell leukemia cell lines transfected with Wls shRNAi. (B) Decrease of Wls expression in Reh cells infected with Wls shRNAi was showed to abrogate canonical Wnt signaling induced by Wnt-3a stimulation. (C) Inhibition of c-Myc, cyclin D1 and Skp2 expression induced by Wnt-1 and Wnt-3a in the Reh cells infected with Wntless shRNAi. Arrow head, cyclin D1. (D) Reh cells infected with Wntless shRNAi showed a decrease in pro-inflammatory cytokines (IL-1β, IL-6, IL-8, GM-CSF and cox-2) expression induced by Wnt-3a treatment. Each experiment was repeated at least three. The results are shown as means ± SD. (***p < 0.001).

Article Snippet: The primary antibodies used in this study were β-actin polyclonal antibody (1:2000, Santa Cruz, I-19), cIAP, Bcl-xL polyclonal antibody (1:2000, Santa Cruz), caspase 3, caspase 8, c-Myc (1:1000, Upstate), cyclin D1 (SeroTec.), FITC-conjugated anti-mouse, alkaline phosphatase-conjugated anti-rabbit antibodies (1:500, Jackson ImmunoResearch Lab.) and Wntless antibody (1:2000, Biolegend) (22).

Techniques: Expressing, Activation Assay, Transfection, Infection, Inhibition