Fig 1: 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.
Fig 2: Effect of TGF-β1 on PDC activity and its regulation by phosphorylation. (A) Schematic showing the regulation of PDC activity by protein serine phosphorylation. (B) 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 UUOF. Stain-free imaging of total protein was used to demonstrate equivalent loading. Representative blots of 3 independent experiments. (C) Relative abundance of mRNA transcripts for PDK1, PDK2, PDK3 and PDK4. Results are expressed relative to PDK1 levels and are pooled data from n = 3 experiments. (D) Flow cytometry measurement of protein levels of PDK1, PDK2 and PDK3 in control and TGF-β1-treated cells. (E) Bead-based multiplex assay of total PDH-E1α, phosphoserine (pS) 232, 293 and 300 with and without TGF-β1 treatment. Results in (D, E) are expressed relative to control and are pooled data from n = 3 independent experiments. (F) PDH enzyme activity in whole-cell lysates of vehicle, TGF-β1- and CPI-613-treated UUOF. Measurements were normalised to total protein content and pooled from n = 4 independent experiments. (G) PDH-E1α protein levels and (H) PDC activity in UUOF transiently transfected with plasmids over-expressing wild-type (WT) PDH-E1α or substitution mutants S232A and S232D. Empty vector was used as a control with values expressed relative to control levels and data pooled from n = 3 independent experiments. Plots show mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P-values were determined using unpaired t tests and the two-stage method of Benjamini, Krieger and Yekutieli (FDR = 1%) in (D, E), and using Brown-Forsyth and Welch one-way ANOVA with Dunnett’s T3 multiple comparisons test in (C, F, G, H).
Fig 3: Inhibition of PDK with dichloroacetate (DCA) ameliorates the suppressive effect of TGF-ß1 on PDC activity, acetyl-CoA levels and protein acetylation in UUOF. (A, B) the effect of TGF-ß1 (1 ng/mL), DCA (5 mM) or co-treatment on (A) PDH activity, (B) free acetyl-CoA concentrations, (C) a-SMA levels and (D) pro-collagen I in UUOF. Measurements in (A, B) were normalised to total protein content. (A–D) Plots show mean ± SEM with pooled data from 2–3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P-values were determined using Brown-Forsyth and Welch one-way ANOVA with Dunnett’s T3 multiple comparisons test. (E) Is representative of similar findings from 4 independent experiments probed in parallel.
Fig 4: Divergent effects of TGF-ß1 on PDC transcription in injury-primed and normal renal kidney fibroblasts. (A, B) Ingenuity pathway analysis (IPA) was used to predict the activation state of canonical pathways activated or inhibited by 24-h treatment with 1 ng/mL TGF- ß1 compared to vehicle (PBS) in (A) UUOF or (B) NRKF. IPA uses a literature-based database of known interactions between transcriptional regulators and their target genes to define an expected expression pattern of targets within each pathway for activated (Z-score = 2) or inhibited (Z-score = - 2) states. The P-value provides a measure of how likely the observed association between a specific pathway and each dataset would be if due to chance alone. Adjusted P-value < 0.05 (- log10 = 1.3) were considered significant. Canonical pathways predicted to be activated by TGF-ß1 are shown in red, inhibited pathways are coloured blue with the remainder depicted as open circles. The acetyl-CoA biosynthesis (PDC) pathway is highlighted in yellow. (C, D) Quantitative flow cytometric analysis of TGF-ß1-induced changes in (C) PDH-E1a and (D) PDK1 staining (mean fluorescence intensity, MFI) in UUOF and NRKF after 24 h treatment (pooled data from 3 independent experiments). Plots show mean ± SEM. ***P < 0.001; ****P < 0.0001; ns, not significant. P-values were determined in (C, D) using two-way ANOVA with Sidak’s multiple comparisons test.
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