heatmaps generated by graphpad prism software version 7 for windows  (GraphPad Software Inc)

Journal: International Journal of Molecular Sciences

Article Title: Reorganization of Metabolism during Cardiomyogenesis Implies Time-Specific Signaling Pathway Regulation

doi: 10.3390/ijms22031330

Figure Lengend Snippet: Retinoic X receptor (RXR) heterodimer pathways enriched in in vitro cardiac differentiation. Heatmaps representing the gene expression profiles (log2 RPKM values) of DEGs related to the PPAR/RXR, LXR/RXR, FXR/RXR, and VDR/RXR activation pathways.

Article Snippet: The heatmaps were generated by GraphPad Prism software Version 7 for Windows (GraphPad Software, San Diego, CA, USA).

Techniques: In Vitro, Gene Expression, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Reorganization of Metabolism during Cardiomyogenesis Implies Time-Specific Signaling Pathway Regulation

doi: 10.3390/ijms22031330

Figure Lengend Snippet: Retinoic acid (RA) synthesis and signaling activation during in vitro cardiac differentiation. ( A ) Comparison of DEGs identified in the RA signaling pathway at the mesoderm (D1 × D4) and cardiac progenitor (D4 × D9) commitment stages. ( B ) Fold change values (log) of the DEGs from the D1 × D4, D4 × D9 and D0 × D15 comparisons. ( C ) Heatmaps representing the gene expression profiles (log2 RPKM values) of DEGs related to RA signaling. Red boxes indicate upregulated genes, while green boxes indicate downregulated genes. Rol: retinol, Ral: retinal. Adapted from Ingenuity Pathway Analysis.

Article Snippet: The heatmaps were generated by GraphPad Prism software Version 7 for Windows (GraphPad Software, San Diego, CA, USA).

Techniques: Activation Assay, In Vitro, Comparison, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Reorganization of Metabolism during Cardiomyogenesis Implies Time-Specific Signaling Pathway Regulation

doi: 10.3390/ijms22031330

Figure Lengend Snippet: Lipid metabolism-related genes regulated in in vitro cardiac differentiation. ( A ) Comparison of genes identified in the “phospholipase” canonical pathway at D1 × D4 and D4 × D9. Venn diagram showing the number of DEGs. ( B ) Scheme of phosphatidic acid (PA) metabolism (Adapted from ). ( C ) Heatmaps representing the gene expression profiles (log2 RPKM values) of DEGs related to PA metabolism.

Article Snippet: The heatmaps were generated by GraphPad Prism software Version 7 for Windows (GraphPad Software, San Diego, CA, USA).

Techniques: In Vitro, Comparison, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Reorganization of Metabolism during Cardiomyogenesis Implies Time-Specific Signaling Pathway Regulation

doi: 10.3390/ijms22031330

Figure Lengend Snippet: Biological processes that are highly regulated during in vitro cardiac differentiation. Comparison of DEGs enriched for terms related to “Transport of ion”, “Carbohydrate” and “Secretion of molecules” at different time points. Venn diagrams show the number of DEGs, and the heatmaps represent the gene expression profiles (log2 RPKM values) of common DEGs identified in each comparison.

Article Snippet: The heatmaps were generated by GraphPad Prism software Version 7 for Windows (GraphPad Software, San Diego, CA, USA).

Techniques: In Vitro, Comparison, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: BETP degradation simultaneously targets acute myelogenous leukemic stem cells and the microenvironment

doi: 10.1172/JCI120654

Figure Lengend Snippet: (A) OCI-AML3 (normal or hypoxia-adapted) cells were cultured with or without a monolayer of NMSCs and treated with ARV-825 (50 nM) or cytarabine (1 μM) under either normoxic or hypoxic conditions for 72 hours, and apoptosis was assessed by annexin V assay using flow cytometry (n = 3). (B) OCI-AML3 cells were treated with ARV-825 (10 nM) for 12 or 24 hours and subjected to CyTOF, and a heatmap was generated using the publicly available Broad Institute Gene Pattern Heatmap viewer. OCI-AML3 cells treated for 24 hours were stained for CXCR4 with or without permeabilization to quantify the changes in intracellular or surface CXCR4 expression using (C) a PE-conjugated antibody for the flow-based assay (n = 3) and (D) a AF594-conjugated secondary antibody for confocal imaging (original magnification, ×40). (E) OCI-AML3 cells were treated with ARV-825 (10 nM) or plerixafor (100 nM) (positive control) for 24 hours and subjected to a migration assay 4 hours after incubation in media containing SDF-1 (100 ng). Surface expression of CXCR4 was measured by flow cytometry, and cell migration was measured by collecting the cells from the lower chamber containing SDF-1 following Beckman Vi-CELL counts (n = 3). Whole-cell lysates obtained from OCI-AML3 cells treated with or without ARV-825 in the presence of SDF-1 were processed for immunoblotting with the indicated antibodies. β-Actin served as a loading control. (F) OCI-AML3 cells were treated with ARV-825 (10 nM) for the indicated durations, and whole-cell lysates were analyzed with the indicated antibodies (β-actin served as a loading control). Numbers indicate normalized intensity. PIM1-overexpressing OCI-AML3 cells were treated with ARV-825 (10 nM) for 24 hours, and then (G) whole-cell lysates were analyzed by immunoblotting with the indicated antibodies (α-tubulin was used as a loading control). (H) Surface expression of CXCR4 was analyzed by flow cytometry followed by a migration assay (n = 3). **P < 0.01 and ***P < 0.001, by standard Student’s t test.

Article Snippet: Myc activity, Wnt/β-catenin/Notch, cell-cycle/apoptosis, and PI3K/AKT/mTOR pathways, and target inhibition–, and tumor microenvironment–related protein expression levels in BM cells from vehicle- and ARV-825–treated mice were determined and quantified in LSCs (CD34 + CD38 – CD90 – CD45RA + ) as cluster 1 and depicted as a heatmap generated with GraphPad Prism 7 on the basis of the percentiles of intensities with respect to the vehicle (bottom).

Techniques: Cell Culture, Annexin V Assay, Flow Cytometry, Generated, Staining, Expressing, Imaging, Positive Control, Migration, Incubation, Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: BETP degradation simultaneously targets acute myelogenous leukemic stem cells and the microenvironment

doi: 10.1172/JCI120654

Figure Lengend Snippet: (A) OCI-AML3 and primary AML cells were treated with ARV-825 (10 nM) for 24 hours. GEP analysis was performed on the isolated RNA (n = 3). (A) GSEA was performed on Illumina GEP data and revealed high enrichment (normalized enrichment score [NES] >3) for several gene sets representing downregulation of Myc target genes and Wnt/B catenin pathways, as well as oncogenic pathways and gene sets for cell cycle, hypoxia, metabolism, and Notch). Validation of Wnt/β-catenin pathway downregulation was done by qPCR analysis of the pathway targets AXIN-2 and FRA-1 at 12 and 24 hours (n = 3 independent samples). Statistical significance was calculated using Bonferroni’s method, and adjusted P values were determined. NMSCs were treated with ARV-825 (25 nM) for 24 hours. (B) Representative GSEA found high enrichment for several gene sets representing Myc target genes and oncogenic pathways, as well as cell-cycle, metabolic, hypoxia, oxidative phosphorylation, and Notch gene sets, and GEP of MSCs showed a significant reduction in surface adhesion and SDF-1 expression. w.r.t., with respect to. (C) MSCs treated with DMSO or ARV-825 (25 nM for 24 h) cocultured with OCI-AML3 cells treated with DMSO or ARV-825 (10 nM) for 24 hours and subjected to either determination of ROS using an ENZ-51011 kit (bottom) or whole-cell lysates from AML cells were subjected to immunoblotting with specific antibodies. Tubulin was used as a loading control. (D) GSE76009 data were curated, and GSEA revealed high enrichment (NES >3) of LSC fractions for several gene sets representing hematopoietic stems cells or LSCs (data not shown) and for this gene set of a subgroup of genes regulated by Myc. Illumina GEP data on primary AML cells treated with ARV-825 for 24 hours showed a significant reduction in the number of genes of the 17-gene signature associated with stemness in AML. (E) BM cells were collected from vehicle- and ARV-825–treated mice with AML-PDX and subjected to CyTOF, and data were analyzed using SPADE 3.0. The tree was generated according to the expression of CD34, CD38, CD90, and CD45RA (top). Myc activity, Wnt/β-catenin/Notch, cell-cycle/apoptosis, and PI3K/AKT/mTOR pathways, and target inhibition–, and tumor microenvironment–related protein expression levels in BM cells from vehicle- and ARV-825–treated mice were determined and quantified in LSCs (CD34+CD38–CD90–CD45RA+) as cluster 1 and depicted as a heatmap generated with GraphPad Prism 7 on the basis of the percentiles of intensities with respect to the vehicle (bottom). UP, upregulated; DN, downregulated.

Article Snippet: Myc activity, Wnt/β-catenin/Notch, cell-cycle/apoptosis, and PI3K/AKT/mTOR pathways, and target inhibition–, and tumor microenvironment–related protein expression levels in BM cells from vehicle- and ARV-825–treated mice were determined and quantified in LSCs (CD34 + CD38 – CD90 – CD45RA + ) as cluster 1 and depicted as a heatmap generated with GraphPad Prism 7 on the basis of the percentiles of intensities with respect to the vehicle (bottom).

Techniques: Isolation, Biomarker Discovery, Phospho-proteomics, Expressing, Western Blot, Control, Generated, Activity Assay, Inhibition