Journal: International Journal of Molecular Sciences

Article Title: “Betwixt Mine Eye and Heart a League Is Took”: The Progress of Induced Pluripotent Stem-Cell-Based Models of Dystrophin-Associated Cardiomyopathy

doi: 10.3390/ijms21196997

Figure Lengend Snippet: Principal studies using cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) to model and study dystrophin-associated cardiomyopathy (DAC).

Article Snippet: A testament to the translatability of this research group’s approach constitutes the results of the halt cardiomyopathy progression in Duchenne “HOPE” randomized, open-label, interventional phase I/II clinical trial (NCT02485938) of intracoronary delivery of an allogeneic CDC cell-therapy product (CAP-1002, Capricor Therapeutics™, Beverly Hills CA, USA) in 25 DMD patients (males >12 years of age, genetically diagnosed with DMD, with significant cardiac scarring).

Techniques: Derivative Assay, Mutagenesis, Expressing, Generated, HAC Assay, Sequencing, Activation Assay, Inhibition, CRISPR, Functional Assay, Activity Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Single-cell transcriptomics reveals striking heterogeneity and functional organization of dendritic and monocytic cells in the bovine mesenteric lymph node

doi: 10.3389/fimmu.2022.1099357

Figure Lengend Snippet: Dendritic cells. (A) Visualization of key genes in feature plots to identify clusters containing total DCs ( FLT3 ), cDC1 ( ANPEP , cluster 1), cDC2 ( FCER1A , clusters 5 and 9) and pDC ( CD4 , cluster 17). (B) Dot plot of key subset-defining genes in selected DC clusters. (C) Heatmap of the top 20 (p_adj) differentially expressed genes in clusters identified as cDC1 (c1), cDC2 (c5 & c9) and pDC (c17). (D) Top genes differentially expressed between cDC2 clusters c5 and c9, visualized as violin plots (linear y-axis). (E–G) Expression of selected signature genes for DC clusters that could not be clearly assigned to a DC subset based on key gene expression.

Article Snippet: Most notably Galectin-1 ( LGALS1 ), which was highly expressed in both resident and migratory cDC1 (c1, c6) and in inflammatory and migratory cDC2 (c3, c8).

Techniques: Expressing, Gene Expression

Journal: Frontiers in Immunology

Article Title: Single-cell transcriptomics reveals striking heterogeneity and functional organization of dendritic and monocytic cells in the bovine mesenteric lymph node

doi: 10.3389/fimmu.2022.1099357

Figure Lengend Snippet: Dendritic cells and monocytes in cluster 3. (A) Expression of DC-associated and monocyte-associated genes in cluster 3. (B) Heatmap of the top 10 (p_adj) differentially expressed genes between cluster 3 and the cDC2 clusters c5 & c9. (C) Visualization of selected genes differentially expressed between c3, c5 and c9. Arrow indicates cluster 3. (D) Heatmap of the top 10 (p_adj) differentially expressed genes between cluster 3 and the monocytic cluster 2. (E) Visualization of selected genes in feature plots showing c2, c3, c5, and c9. (F) Proposed differentiation pathways of inflammatory cDC2 (c11→c9→c5→c3), moDC (c2→c3) and DC3 (c11→c3) indicated by dashed arrows. Arrows in feature plots indicate location of putative DC3 progenitors.

Article Snippet: Most notably Galectin-1 ( LGALS1 ), which was highly expressed in both resident and migratory cDC1 (c1, c6) and in inflammatory and migratory cDC2 (c3, c8).

Techniques: Expressing

Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: Cdc7 depletion in IMR90 fibroblasts causes cell cycle arrest in G1. (A) Time course of CDC7 mRNA knock-down (KD) in IMR90 cells relative to cells transfected with control-siRNA (CO). (B) Whole cell extracts (WCE) prepared from untreated (UT), CO and Cdc7KD cells were analysed by immunoblotting with the indicated antibodies (β-actin—loading control). (C) At the indicated time points, cell number was measured in UT, CO and Cdc7KD cell populations. (D) DNA content of UT, CO and Cdc7KD cells at 96 and 120 h post-transfection. (E) DNA content of double thymidine-arrested cells (DTB), CO and Cdc7KD cells 48 h after release from double thymidine block and transfection, and asynchronous Cdc7KD cells 48 h post-transfection. (F) At 96 h post-transfection, cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody. DNA was stained with propidium iodide (PI). Numbers show the percentage of cells incorporating BrdU.

Article Snippet: Oligos CDC7-B and CDC7-A showed comparable gene silencing efficacy (mRNA and protein reduction) and induced similar phenotypic effects (accumulation of cells with G1 DNA content) ( ; Supplementary Figure 2A and B ).

Techniques: Transfection, Western Blot, Blocking Assay, Staining

Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: CDK activity and replication initiation factors are downregulated in response to Cdc7 depletion. (A) WCE prepared from IMR90 cells transfected with control-siRNA (CO) and CDC7-siRNA (Cdc7KD) were analysed by immunoblotting with the indicated antibodies (β-actin—loading control). (B) WCE prepared from CO and Cdc7KD cell populations and from CO cells treated with 200 μM roscovitine (ROS) for 24 h (negative control) and MDA-MB231 breast cancer cells (positive control) were immunoprecipitated with anti-cyclin A and anti-cyclin E antibodies. Cdk2 immunoprecipitates (Cdk2 IP) were subjected to an in vitro kinase assay using recombinant truncated Rb protein (p56) as substrate. In vitro phosphorylation was detected with a specific anti-Rb-phospho-Thr-821 antibody. Note that lanes 1–8 were run on the same polyacrylamide gel and proteins transferred to the same PVDF membrane by semi-dry electroblotting. The membrane was subsequently cut as indicated for optimized immunodetection. (C) WCE and chromatin-bound protein fractions (CBF) prepared from untreated (UT), CO and Cdc7KD cells (96 h post-transfection) were analysed by immunoblotting with the indicated antibodies (β-actin and histone H1—loading controls). (D) WCE from UT, CO and Cdc7KD cells (48 and 96 h post-transfection) were analysed by western blotting with the indicated antibodies (β-actin—loading control). (E) WCE from UT, CO and Cdc7KD cells (48 and 96 h post-transfection) and from cells treated for 24 h with 17 μM cisplatin (Pt) were analysed by western blotting with the indicated antibodies (β-actin—loading control).

Article Snippet: Oligos CDC7-B and CDC7-A showed comparable gene silencing efficacy (mRNA and protein reduction) and induced similar phenotypic effects (accumulation of cells with G1 DNA content) ( ; Supplementary Figure 2A and B ).

Techniques: Activity Assay, Transfection, Western Blot, Negative Control, Positive Control, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Immunodetection

Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: p53-dependent upregulation of Wnt/β-catenin signalling antagonist Dkk3 is required for Cdc7-depletion-induced cell cycle arrest. (A) Cytoplasmatic protein fractions (CF) and crude nuclear extracts (NE) from untreated (UT), control-siRNA (CO) and CDC7-siRNA (Cdc7KD)-transfected IMR90 cells (72 h post-transfection) were analysed by immunoblotting with the indicated antibodies (β-actin and Orc4—loading controls). (B) At the same time point, CO and Cdc7KD cells were fixed and β-catenin, Myc and cyclin D1 detected by indirect immunofluorescence using a fluorescein-labelled secondary antibody. (C, D) CF and NE samples prepared from CO, Cdc7KD and doubly depleted Cdc7/Dkk3 (Cdc7KD/Dkk3KD) cells 48 and 72 h post-transfection were analysed by immunoblotting with the indicated antibodies. (E) 72 h post-transfection CO, Cdc7KD and Cdc7KD/Dkk3KD cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody. Chromatin-bound PCNA and γH2A.X (inset: higher magnification) were detected by indirect immunofluorescence with anti-PCNA and anti-γH2A.X antibodies and a fluorescein-labelled secondary antibody. DNA was stained with propidium iodide (BrdU) or DAPI (PCNA and γH2A.X). Apoptotic cell death was detected in doubly depleted Cdc7KD/Dkk3KD cells by (F) phase contrast microscopy and by (G) immunoblot analysis of CF and NE with the indicated antibodies (β-actin and Orc4—loading controls).

Article Snippet: Oligos CDC7-B and CDC7-A showed comparable gene silencing efficacy (mRNA and protein reduction) and induced similar phenotypic effects (accumulation of cells with G1 DNA content) ( ; Supplementary Figure 2A and B ).

Techniques: Transfection, Western Blot, Immunofluorescence, Staining, Microscopy

Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: FoxO3a-mediated upregulation of ARF and CDK inhibitors is an essential step for arresting cell cycle progression in Cdc7-depleted cells. (A) Cytoplasmatic protein fractions (CF) and crude nuclear extracts (NE) prepared from CO, FoxO3aKD, Cdc7KD and doubly depleted Cdc7/FoxO3a (Cdc7KD/FoxO3aKD) IMR90 cells 48 h post-transfection were analysed by immunoblotting with the indicated antibodies (β-actin and Orc4—loading controls). Note that the faster migrating band represents the hypo-phosphorylated, active form of p27 and the slower migrating band its hyper-phosphorylated inactive form (Rodier et al, 2001; Chopra et al, 2002) (B) DNA content of Cdc7KD and Cdc7KD/FoxO3aKD cells 48 h post-transfection. (C) 48 h post-transfection CO, Cdc7KD and Cdc7KD/FoxO3aKD cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody (inset: higher magnification). Chromatin-bound PCNA and γH2A.X were detected as described in the legend to Figure 3. Apoptotic cell death was detected 48 h post-transfection in doubly depleted Cdc7KD/FoxO3aKD cells (D) by phase contrast microscopy and (E) by immunoblot analysis of CF and NE with the indicated antibodies (β-actin and Orc4—loading controls).

Article Snippet: Oligos CDC7-B and CDC7-A showed comparable gene silencing efficacy (mRNA and protein reduction) and induced similar phenotypic effects (accumulation of cells with G1 DNA content) ( ; Supplementary Figure 2A and B ).

Techniques: Transfection, Western Blot, Microscopy

Journal: The EMBO Journal

Article Title: Molecular architecture of the DNA replication origin activation checkpoint

doi: 10.1038/emboj.2010.201

Figure Lengend Snippet: The Cdc7-depletion-induced cell cycle arrest is p15INK4B dependent. (A) Upregulation of p15 levels in Cdc7-depleted IMR90 cells was confirmed by immunoblotting WCE prepared from CO cells and Cdc7-depleted cells 48, 96 and 120 h post-transfection with antibodies against p15 and β-actin (loading control). (B) Cdc7KD and CO cells were fixed 96 h post-transfection and p15 detected by indirect immunofluorescence using a fluorescein-labelled secondary antibody. (C) CF and NE prepared from CO, Cdc7KD and doubly depleted Cdc7/p15 (Cdc7KD/p15KD) cells 72 h post-transfection were analysed by immunoblotting with the indicated antibodies (β-actin and Orc4—loading controls). (D) 72 h post-transfection CO, Cdc7KD and Cdc7KD/p15KD cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody. Chromatin-bound PCNA and γH2A.X were detected as described in Figure 3 legend. Apoptotic cell death was detected 72 h post-transfection in doubly depleted Cdc7KD/p15KD cells by (E) phase contrast microscopy and by (F) immunoblot analysis of CF and NE with the indicated antibodies (β-actin and Orc4—loading controls).

Article Snippet: Oligos CDC7-B and CDC7-A showed comparable gene silencing efficacy (mRNA and protein reduction) and induced similar phenotypic effects (accumulation of cells with G1 DNA content) ( ; Supplementary Figure 2A and B ).

Techniques: Western Blot, Transfection, Immunofluorescence, Microscopy