Journal: PLoS ONE

Article Title: Modelling the developmental spliceosomal craniofacial disorder Burn-McKeown syndrome using induced pluripotent stem cells

doi: 10.1371/journal.pone.0233582

Figure Lengend Snippet: For the analyses, data from two patient iPSC lines, two parent iPSC lines and two unrelated control iPSC lines were pooled. A) Relative TXNL4A mRNA expression levels for patient iPSCs compared to mother iPSCs and unrelated control iPSCs, determined using qPCR of cDNA from each cell line. Graphs were obtained using the ΔΔC T method with ACTB as the endogenous reference gene and normalised to the unrelated control line SW171A. n = 4. B) Relative proliferation for patient iPSCs compared to mother iPSCs and unrelated control iPSCs using an MTT assay to monitor cell proliferation, normalised to the KW191A maternal line. n = 4. C) Percentages of apoptotic, necrotic and live cells for patient, mother and unrelated control iPSCs determined using co-staining with annexin-V and DAPI then quantification of cell subpopulations by flow cytometry. n = 3. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

Article Snippet: Cells were washed once in PBS and then washed once in 1X Annexin-V Binding Buffer (ABB) (Invitrogen).

Techniques: Expressing, MTT Assay, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Modelling the developmental spliceosomal craniofacial disorder Burn-McKeown syndrome using induced pluripotent stem cells

doi: 10.1371/journal.pone.0233582

Figure Lengend Snippet: A) Percentages of apoptotic, necrotic and live cells for patient, mother and unrelated control iNCCs determined using co-staining with annexin-V and DAPI and quantification of cell subpopulations by flow cytometry. Data from the two patient lines, the two mother lines and the two unrelated control lines was pooled for analysis. n = 3. B) Principal Component Analysis (PCA) for all iPSC lines from RNA-Seq performed on all iNCC lines. Whole transcriptome RNA-Seq analysis was performed on triplicate samples of RNA isolated from all six iNCC lines. For analysis, data from the patient lines was pooled and data from the mother and unrelated control lines was pooled (pooled controls). C) Key marker gene expression levels in iNCCs from RNA-Seq data. Mean normalised read counts for AXIN2 , neural crest and neural border marker genes. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

Article Snippet: Cells were washed once in PBS and then washed once in 1X Annexin-V Binding Buffer (ABB) (Invitrogen).

Techniques: Staining, Flow Cytometry, RNA Sequencing Assay, Isolation, Marker, Expressing

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 4. The effects of milk-derived extracellular vesicles (MEVs) supplementation on baseline homeostatic human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148-5P transcript levels (a), DNMT1 protein abundance (b) and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (d), and DNMT enzymatic activity and DNMT1 protein level (e). * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 6. The effects of milk-derived extracellular vesicles (MEVs) on IFN-γ primed human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (p < 0.05) (d), and DNMT enzymatic activity and DNMT1 protein level (p < 0.05) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 7. The effects of milk-derived extracellular vesicles (MEVs) on primed human microglia clone 3 (HMC3) cells 9 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels relative to miR-148a-5P levels (p ≤ 0.05) (d), and DNMT enzymatic activity relative to DNMT1 protein level (A.U) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control