Extended Data Fig. 1: TGFβ-induced collagen synthesis is linked to glutamine-dependent TCA cycle anaplerosis.
From: Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment
(A) Growth curves of PSCs cultured in the indicated percentage of Gln and treated with TGFβ (2 ng/mL). n = 3 biologically independent samples. (B) Western Blot of PSCs cultured in 100% or 20% Gln and treated with TGFβ for 48 h. (C) Collagen abundance in ECM derived from confluent PSCs cultured in the indicated percentage of Gln and treated with TGFβ. n = 3 biologically independent samples. (D) Growth curves of MFBs cultured in the indicated percentage of Gln and treated with TGFβ. n = 3 biologically independent samples. (E) Western Blot of MFBs cultured in 100% or 20% Gln and treated with TGFβ for 48 h. (F) Collagen abundance in ECM derived from confluent MFBs cultured in the indicated percentage of Gln and treated with TGFβ. n = 3 biologically independent samples. (G) Relative number of NIH-3T3 cells cultured in 10% Gln and treated with TGFβ and 0.2, 1 or 5 mM of asparagine (Asn) or proline (Pro). n = 3 biologically independent samples. (H) Western Blot of NIH-3T3 cells cultured in 10% Gln and treated with TGFβ and the indicated metabolites and concentrations for 48 h. (I) Relative metabolite abundance in NIH-3T3 cells cultured in 10% Gln and treated with TGFβ and dm-Glu (5 mM) or dm-αKG (5 mM) for 48 h. n = 3 biologically independent samples. (J,K) Western Blot of PSCs (J) or MFBs (K) cultured in 100% or 20% Gln and treated with TGFβ for 48 h. (L,M) tRNA charging in PSCs (L) or MFBs (M) cultured in 20% Gln and treated with TGFβ for 48 h. n = 1 independent experiment. (N,O) Western Blot of PSCs (N) or MFBs (O) cultured in 20% Gln and treated with TGFβ and dm-Glu or dm-αKG for 48 h. MFBs were also treated with aspartate (Asp, 20 mM). Mean±SD (A,C,D,F) or mean+SD (G) are shown. Dashed lines (A,D,G) represent cell number at day 0. Two-way ANOVA (A,C,D,F). Western blots are representative of three (B,E,J,K) or two (H,N,O) independent experiments. tRNA charging analyses (L,M) are representative of two independent experiments. All other experiments were performed at least twice.
