Extended Data Fig. 3: TGFβ suppresses PC expression and reduces PC activity.
From: Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment
(A-C) mRNA expression of the indicated genes in NIH-3T3 cells (A), PSCs (B) or MFBs (C) cultured in 100% Gln and treated with TGFβ for 24 h. n = 3 biologically independent samples. (D) Western Blot of PSCs (left) or MFBs (right) cultured in 100% or 20% Gln and treated with TGFβ for 48 h. (E) UCSC genome browser tracks showing putative SMAD2/SMAD3/SMAD4 binding motifs, SMAD4 ChIP-seq peaks in HepG2 cells, the Genehancer promoter element and the PC transcriptional start site (TSS) at the genomic loci of three human PC isoforms. (F) Pcx expression from RNA-sequencing of quiescent PSCs (qPSC), myofibroblastic CAFs (myCAF) and inflammatory CAFs (iCAFs). Data and p-values are from GSE93313. (G) mRNA expression of myCAF and iCAF markers in iCAFs and myCAFs (left); Pcx mRNA expression in myCAFs and iCAFs, relative to qPSCs (right). n = 3 biologically independent samples. (H) [U-13C]-Glc tracing in PSCs cultured in 100% or 20% Gln and treated with TGFβ for 48 h. n = 3 biologically independent samples. (I) [U-13C]-Glc tracing into indicated amino acid residues of cellular proteins. NIH-3T3 cells were cultured in 10% Gln and treated with TGFβ for 48 h. n = 3 biologically independent samples. (J,K) [3,4-13C]-Glc tracing in PSCs cultured in 100% or 20% Gln and treated with TGFβ for 48 h. M + 1 labeling (J). PC activity (K). n = 3 biologically independent samples. (L) Western Blot of PSCs expressing empty vector or human PC cDNA, cultured in 20% Gln and treated with TGFβ. Mean+SD (A-C,G-K) are shown. Two-sided unpaired t-test (A-C,G,I), one-way ANOVA with Holm-Sidak correction (H,J,K). Western blots (D,L) are representative of two independent experiments. [3,4-13C]-Glc tracing in PC-ko cells (F) was performed once. All other experiments were performed at least twice.
