p73  (Bethyl)

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: A Interrogation of ChIP-seq data indicated binding of TAp73α, TAp73β and p53 to the putative OPA1 promoter region. Sequencing read files were obtained from the GEO data set GSE15780, and tracks shown are for the indicated transcription factors at selected genes. B , C Targeted ChIP of TAp73 bound chromatin. RT-qPCR primers were designed in the promoter region of the OPA1 gene (OPA1 ‘A’ and OPA1 ‘B’). Red squares indicate regions enriched for the Trp73 motif (p < 0.001). qPCR was performed to quantify the fold enrichment of the OPA1 promoter region in an IP sample relative to IgG control. Enrichment of MDM2 and SAT2 promoter regions were assayed as positive and negative controls, respectively. qPCR was carried out on 3 independent ChIP experiments and data shown as individual data points ± SD (n = 3). D Representative western blot of mitochondrial fusion proteins in TAp73 KO and WT control. Cells were transfected with either EV or TAp73α expression construct for 24 h. E Densitometry analysis of western blot data shown in (D). Band intensity was calculated for total OPA1 (long and short isoforms) and CDKN1A (positive control). Signal intensity was normalised to loading control and reported relative to WT cells transfected with empty vector (n = 3). *P ≤ 0.05, (n.s.) not significant (Student’s t-test, comparison of indicated condition with WT empty vector control). F RT-qPCR was performed against OPA1 , MFN2 and CDKN1A genes and expression values calculated using the ∆∆Ct method, relative to WT empty vector control. Data shown as mean ± SD (n = 3). *P ≤ 0.05, (n.s) not significant (Student’s t-test, comparison of indicated condition with WT empty vector control).

Article Snippet: TAp73 , Bethyl , A300-126A , Rabbit , 1:1000.

Techniques: ChIP-sequencing, Binding Assay, Sequencing, Quantitative RT-PCR, Control, Western Blot, Transfection, Expressing, Construct, Positive Control, Plasmid Preparation, Comparison

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: A WT or TAp73 KO H1299 cells were transfected with the indicated plasmids for 24 h and IF carried out against ATP5B (green) with DAPI nuclear counterstain (blue). Cells transfected with HA-TAp73α expression plasmid were stained for HA as a transfection control (red). Lower magnification images, scale bar = 10 μm; Callout images, scale bar = 4 μm. B Representative western blot of OPA1 expression following transfection of WT and TAp73 KO cells with pCMW-OPA1 construct. C Quantification of mitochondrial morphology from ( A ) using Zeiss Intellesis module, trained to segment individual mitochondria. Statistical significance for each condition was calculated using Student’s t-test, comparing with WT EV control (column 1); *p < 0.05, (ns) not significant (n = 3). D Transmission electron micrographs of mitochondrial morphology from WT and TAp73 KO cells. Scale bar = 100 nm. E Mitochondrial length measurements obtained from ( D ). ****p < 0.0001 in Student’s t-test. A minimum of 100 mitochondria were measured from n = 3 independent biological replicates. F , G Mitochondrial stress test performed on Seahorse XFe96 analyser. Canonical mitochondrial inhibitors injected sequentially as labelled (Oligomycin = 2 μM, FCCP = 500 nM, Antimycin A/ Rotenone = 2 μM). The indicated mitochondrial stress test parameters were calculated from OCR data. Data were corrected for non-mitochondrial OCR, normalised to cell number, and are shown as mean ± SD (n = 3). *P ≤ 0.05 and **P ≤ 0.01 in Student’s t-test relative to WT control. H Western blot of the indicated ETC subunits in wild-type and TAp73 KO cells, obtained using OXPHOS antibody cocktail. I qPCR against mt-CO2 , expressed relative to expression of nuclear encoded β2-microglobulin . Relative expression was calculated using the ΔΔCt method and expressed as a percentage of wild-type control (n = 2 biological replicates, each tested in triplicate). n.s not significant in Student’s t-test.

Article Snippet: TAp73 , Bethyl , A300-126A , Rabbit , 1:1000.

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Control, Western Blot, Construct, Transmission Assay, Injection

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: A Kinetics of apoptosis induction was tracked using AnnexinV/FITC dye following treatment with BH3-mimetic. Wild-type or TAp73 KO H1299 cells were treated with combination treatment of ABT-737 and S63845 at the indicated concentrations. Data points plotted as mean ± SD from n = 6 technical replicates. *P ≤ 0.05 (Student’s t-test), when comparing Cas9 control with TAp73 KO cells at 90 min and at the indicated dose of BH3-mimetic (0.5 µM and 1 µM). B Representative western blot against pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family in WT and TAp73 KO cell lines. C Representative TEM micrographs of mitochondrial ultrastructure in WT and TAp73 KO cells. Yellow arrows highlight regions of disorganisation or loss of cristae in TAp73 KO cells. Scale bar = 200 nm. Quantification of mitochondrial cristae width ( D ) and cristae density ( E ) in TEM micrographs obtained from WT and TAp73 KO H1299 cells. Measurements were obtained from n ≥ 50 mitochondria, across two biological replicates. ****p < 0.0001 in Student’s t-test.

Article Snippet: TAp73 , Bethyl , A300-126A , Rabbit , 1:1000.

Techniques: Control, Western Blot

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: TAp73 expression is required for mitochondrial homeostasis in vitro and in the ciliated epithelium in vivo (green nuclei) (left). Conversely, TAp73 ablation leads to decreased OPA1 expression, mitochondrial fission, and impaired mitochondrial function (right). The Trp73 −/− tracheal epithelium maintains a limited expression of FOXJ1 positive cells (purple), indicating additional mechanisms, such as the observed mitochondrial dysfunction drives MCC loss and COPD pathogenesis.

Article Snippet: TAp73 , Bethyl , A300-126A , Rabbit , 1:1000.

Techniques: Expressing, In Vitro, In Vivo

Journal: Cell Death & Disease

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1038/s41419-024-07130-6

Figure Lengend Snippet: A list of antibodies used in Western Blotting experiments.

Article Snippet: TAp73 , Bethyl , A300-126A , Rabbit , 1:1000.

Techniques: Western Blot