Journal: Cell reports

Article Title: Piwil1 Regulates Glioma Stem Cell Maintenance and Glioblastoma Progression

doi: 10.1016/j.celrep.2020.108522

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-CCND2 , Novus Biologicals , Cat# NBP2-14460.

Techniques: Recombinant, Staining, Imaging, Membrane, Cell Culture, SYBR Green Assay, Plasmid Preparation, shRNA, Software

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: Molecular Biology of the Cell

Article Title: Cardiac inducing colonies halt fibroblast activation and induce cardiac/endothelial cells to move and expand via paracrine signaling

doi: 10.1091/mbc.e22-02-0032

Figure Lengend Snippet: FIGURE 6: HCM and HCF cell cycle activities were analyzed in response to rPF cocktail or CiC-CM. (A–C) HCMs show increased EdU labeling when cultured in CiC-CM. (A) EdU labeling in HCMs after 30 min of culture. Arrows point to a few representative positive nuclei (top left panel). DAPI and Edu merge confirms nuclear staining (A, top middle) while phase contrast image (A, top right) shows healthy cells. (B, C) EdU staining in HCMs first incubated in CiC-CM from a 31 YO (B) or a 66 YO (C) individual. Both show increased EdU staining (arrows), which was quantified and graphed (D). There are significantly more EdU-positive nuclei in CiC-CM–treated HCMs. (E–K) The cyclin D2+ nuclei from F–K were counted from the 500 µm area for both treatments. Cyclin D2 was analyzed in HCMs moving into a myocardial wound model using immunofluorescence after culturing for 24 h in CiC-CM or control DMEM. (F–H) Few yclin D2–positive cells (red) are present after 24 h within the 500 µm region in control DMEM treatment. (I–K) Twenty-four hours after CiC-CM treatment, CycD2- positive cells are markedly more observable. The graph in E shows that the number of cyclinD2+ nuclei was significantly higher in CiC-CM treatment than control DMEM treatment. (L) qPCR analyses were done on untreated HCMs, 24 h CiC-CM HCMs, and 24 h CiC-CM 24 h no CiC-CM HCMs probing for CDK-1 (blue), cyclin D1 (orange), and cyclin B1 (gray) genes. Gene expression levels were plotted relative to GAPDH, and expression levels seen in untreated HCMs were normalized to 1. N = 3 for the experiment represented in this figure.

Article Snippet: Primary antibodies: Most the same as for Western blots (above), others: cyclin D2 (Novus Biologicals Cat #NBP2-14460-25 ul), phalloidin (Life Technologies Cat #R415), vinculin (Proteintech Cat #66305-1-1g), Rac-GTP (NewEast Biosciences Cat #26903), EdU (and PCNA) (Santacruz Biotech Cat #sc-56).

Techniques: Labeling, Cell Culture, Staining, Incubation, Immunofluorescence, Control, Gene Expression, Expressing

Journal: International Journal of Molecular Sciences

Article Title: miR-198 Represses the Proliferation of HaCaT Cells by Targeting Cyclin D2

doi: 10.3390/ijms160817018

Figure Lengend Snippet: MiR-198 directly bound to the 3′-UTR of CCND2 mRNA. ( A ) Bioinformatics analyses showed that 10 potential target genes of miR-198 were predicted by three different databases; ( B ) The two distinct predicted binding sites of miR-198 in the 3′-UTR of CCND2 mRNA were allocated, and the fragments containing either mutated binding site were amplified according to the mature miR-198 sequence; ( C ) In pMIR-REPORT™ vector, CCND2 mRNA 3′-UTRfragment containing either the wild type or the mutated Site 1 was fused downstream the reporter gene. When the vectors were cotransfected with miR-198 mimic or mimic control, and the relative luciferase activity, normalised by β-gal, was significantly suppressed in vector with wild type Site 1 than that with mutated Site 1 (32.80% ± 6.89%); ( D ) In the presence of wild type Site 2, not the mutated one, miR-198 was able to significantly inhibit luciferase activity although to a less extent than wild type Site 1 (55.39% ± 8.48%). (NC, negative control; WT, wide-type; MT, mutated-type. * Compared with NC, p < 0.05).

Article Snippet: The antibodies used were as follows: mouse anti-GAPDH (AG019, Beyotime, China), mouse anti-CCND2 (BA2347-2, BOSTER, Wuhan, China), Horse Radish Peroxidase (HRP)-abeled Goat Anti-Mouse Immunoglobulin G (IgG) (H + L) (A0216, Beyotime, China).

Techniques: Binding Assay, Amplification, Sequencing, Plasmid Preparation, Luciferase, Activity Assay, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: miR-198 Represses the Proliferation of HaCaT Cells by Targeting Cyclin D2

doi: 10.3390/ijms160817018

Figure Lengend Snippet: MiR-198 transfection repressed the mRNA and protein expression of CCND2. ( A ) MiR-198 mimic transfection reduced the expression of CCND2 mRNA at 24 h (68.09% ± 16.73%) and 48 h (45.68% ± 10.94%); ( B ) MiR-198 mimic or CCND2 siRNA transfection reduced the expression of CCND2 protein at 24 h (50.55% ± 24.04% or 34.45% ± 6.98%) and 48 h (17.38% ± 9.96% or 16.24% ± 9.23%). (NC, negative control. * Compared with NC, p < 0.05).

Article Snippet: The antibodies used were as follows: mouse anti-GAPDH (AG019, Beyotime, China), mouse anti-CCND2 (BA2347-2, BOSTER, Wuhan, China), Horse Radish Peroxidase (HRP)-abeled Goat Anti-Mouse Immunoglobulin G (IgG) (H + L) (A0216, Beyotime, China).

Techniques: Transfection, Expressing, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: miR-198 Represses the Proliferation of HaCaT Cells by Targeting Cyclin D2

doi: 10.3390/ijms160817018

Figure Lengend Snippet: CCND2 siRNA transfection inhibited HaCaT cell proliferation by blocking cell cycle at G1 phase. ( A ) CCND2 siRNA transfection led to a significant reduction of mRNA expression of CCND2 in HaCaT cells both at 24 h (73.79% ± 11.45%) and 48 h (51.18% ± 8.85%); ( B ) Cell viability analysis showed that CCND2 siRNA transfection inhibited HaCaT cell proliferation at 24 h (67.98% ± 7.31%) and 48 h (67.45% ± 6.70%); ( C ) FCM analysis showed the effect of CCND2 siRNA transfection on cell cycle progression, and G1 phase arrest was obvious at 24 h (55.51% ± 6.18%) and 48 h (70.19% ± 4.10%) compared with negative control (42.98% ± 2.48%). (NC, negative control. * Compared with NC, p < 0.05).

Article Snippet: The antibodies used were as follows: mouse anti-GAPDH (AG019, Beyotime, China), mouse anti-CCND2 (BA2347-2, BOSTER, Wuhan, China), Horse Radish Peroxidase (HRP)-abeled Goat Anti-Mouse Immunoglobulin G (IgG) (H + L) (A0216, Beyotime, China).

Techniques: Transfection, Blocking Assay, Expressing, Negative Control