Fig. 7. PDK2 regulates PDH activity and acetyl-CoA production in CAFs.

a, Predicted kinase activity in cCAFs compared to cNFs based on the modelling of their MS-based phosphoproteomic data. b,c, Representative western blots (b) and quantification (c) showing PDHA1 phosphorylation levels at the regulatory site S293 in mammary NFs and CAFs. VCL was used as a loading control. n = 3 or 4 biological replicates. d, Pyruvate dehydrogenase activity of NFs and CAFs measured as the rate of NAD+ reduction in vitro. n = 3–6 biological replicates. e, Representative western blot showing PDK2 levels in paired mammary NFs and CAFs. VCL was used as a loading control. f, PDK1–4 expression in mammary NFs and CAFs in culture, measured by qPCR and normalized to 18S expression. n = 4 biological replicates. g, PDK1–4 mRNA expression in LCMD sections of normal and TNBC-associated stroma from Saleh et al.³¹. h,i, Representative western blot (h) and quantification (i) showing PDHA1 phosphorylation levels in cNFs transfected with siCtl or siPDK1–4. n = 3 biological replicates. VCL was used as a loading control. j, Intracellular acetyl-CoA unlabelled (black) and ¹³C₂-labelled (coloured) from ¹³C₆-glucose measured by MS in cNFs transfected with siCtl or siPDK2. n = 3 biological replicates. k, Representative western blot showing PDHA1 phosphorylation levels in cCAFs transfected with empty vector, pGC-PDK2^N255A or pGC-PDK2^WT. l, ¹³C₂-labelled (coloured bar) and unlabelled (black bar) acetyl-CoA measured by MS in cCAFs transfected with empty vector, pGC-PDK2^N255A or pGC-PDK2^WT and labelled with ¹³C₆-glucose. n = 3 biological replicates. Error bars indicate mean ± s.e.m. P values were calculated with two-tailed, unpaired t-test with Welch’s correction (g and j), two-tailed Mann–Whitney test (c and d), two-way ANOVA with Tukey’s multiple comparisons test (f), one-way ANOVA with Dunnett’s multiple comparison test (l) or Kruskal–Wallis with Dunn’s multiple comparison test (i).

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