Figure 4
From: TGF-β1 is a regulator of the pyruvate dehydrogenase complex in fibroblasts
Effect of TGF-β1 on the compartmentalisation of PDH in UUOF. (A) Representative immunofluorescent staining of PDH-E1α (green) in UUOF with and without treatment with exogenous TGF-β1 (1 ng/mL) for 24 h. Nuclei were stained with DAPI (blue). Bar = 20 µm. (B) Representative high-power axial and frontal planes of nuclei from each experimental group. Line plots beneath show PDH-E1α (green) and DAPI (blue) staining intensity across a central nuclear plane (yellow arrow) using Fiji Image J. (C) Volume rendered images of cells in (B) showing change in nuclear and peri-nuclear distribution of PDH-E1α (green) in response to TGF-β1. (D) Nuclear co-localisation of PDH-E1α (green) and the nucleolar protein fibrillarin (red) after TGF-β1 treatment shown as individual stains, merged with DAPI (blue) and as a volume-rendered image. Bar = 5 µm. 3D rendered images were generated using Huygens Professional v19.04 (Scientific Volume Imaging; https://svi.nl). (E) Western blots showing the effect of 1 ng/mL TGF-β1 for 24 h on phosphoserine 293, phosphoserine 232 and total PDH-E1α, PDK1, PGK1 and PDP1 in mitochondrial and nuclear subcellular fractions of UUOF shown in Fig. 2B. Tomm20/Cyt C and lamin B/histone H3 were used to demonstrate purity of mitochondrial and nuclear proteins, respectively. Stain-free imaging of total protein was used to demonstrate equivalent loading. Representative blots of 3 independent experiments.
