Journal: Nature Communications
Article Title: Defective vascular smooth muscle cell tafazzin impairs mitochondrial function and promotes atherosclerosis in preclinical models
doi: 10.1038/s41467-025-65873-y
Figure Lengend Snippet: A Putative binding sites of NFκB in the upstream region of the miR-125a-5p transcription start site indicated by MotifMap . TSS = Transcription start site. B NFκB DNA binding activity determined by ELISA using TransAM NFκB p65 Transcription Factor Assay in control hVSMCs (ctrl) or hVSMCs treated with oxidised LDL (ox), oxLDL + Bay 11-7082 (Bay) or oxLDL + DMSO (DMSO) ( n = 5, 2 male (M), 3 female (F) hVSMC donors, 1-way ANOVA, post hoc Bonferroni-Holm). arb. units = arbitrary units. C Representative western blot image for phosphorylated NFκB (P-NFκB) p65, total NFκB p65 (NFκB), tafazzin (Taz), citrate synthase (CS) in control hVSMCs (ctrl) or hVSMCs treated with oxidised LDL (ox), oxLDL + Bay 11-7082 (Bay) or oxLDL + DMSO (DMSO) with quantification. Vinc = vinculin. Data normalised to citrate synthase for tafazzin or vinculin for citrate synthase ( n = 4, 2 M, 2 F, 1-way ANOVA, post hoc Bonferroni-Holm). D Quantitative PCR for miR-125a-5p in control hVSMCs (ctrl) or hVSMCs treated with oxidised LDL (ox), oxLDL + Bay 11-7082 (Bay) or oxLDL + DMSO (DMSO). Expression is shown relative to control, normalised to RNU6-1 ( n = 6, 3 M, 3 F, Kruskal-Wallis test, post-hoc Bonferroni-Holm). E Representative Seahorse profiles of oxygen consumption rate (OCR) with sequential addition of oligomycin (oligo), FCCP and antimycin/rotenone (A/R) in hVSMCs transfected with non-targeting control (ctrl) or miR-125a-5p mimic (mimic) ( n = 3, 3 M). F , G Maximal OCR after FCCP ( n = 6, 3 M, 3 F, two-sided unpaired t test) ( F ) and percentage of cells positive for EdU ( G ) in hVSMCs transfected with control or miR-125a-5p mimic ( n = 5, 3 M, 2 F, two-sided unpaired t test). For ( B–G ) n = number of independent experiments. Data are shown as mean ± SEM with nominal or multiplicity adjusted p - values. Source data are provided as a Source Data file.
Article Snippet: Protein concentration was determined using the BCA method and 4 μg of protein assayed for NFκB p65 DNA binding activity, using the Trans-AM NFκB p65 Transcription Factor Assay kit (Active Motif) according to the manufacturer’s instructions.
Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Transcription Factor Assay, Control, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transfection
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