Extended Data Fig. 5: Fibroblasts take up and use lactate for TCA cycle anaplerosis via PC.
From: Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment
(A) [U-13C]-Glc and [U-13C]-Lac tracing. NIH-3T3 cells were cultured for 48 h in 100% or 10% Gln in the presence of 10 or 1 mM D-glucose with or without 10 mM Na-lactate. M + 3 isotopologues are shown in Fig. 6b. G, glucose; L, lactate. n = 3 biologically independent samples. (B) [U-13C]-Lac tracing. NIH-3T3 cells were cultured in 10% Gln and treated with AZD3965 (MCT1 inhibitor, 5 µM) or sodium oxamate (LDH inhibitor, 10 mM) for 8 h. [U-13C]-Lac was added in the last 1 h. n = 2 (LDHi), n = 3 (all others) biologically independent samples. (C,D) [1-13C]-Lac tracing in NIH-3T3 cells expressing Ctrl or PC sgRNA cultured in 10% Gln in the presence of 10 mM Na-lactate for 48 h. M + 1 labeling (C). PC activity (D). n = 3 biologically independent samples. (E,F) Collagen abundance in ECM generated by confluent parental (E) or Ctrl or PC sgRNA expressing NIH-3T3 cells (F) cultured in 10% Gln and the indicated concentrations of D-glucose and Na-lactate. n = 3 biologically independent samples. (G) Western Blot of PSCs cultured in 20% Gln and the indicated concentrations of D-glucose for 48 h. (H) [U-13C]-Glc and [U-13C]-Lac tracing into indicated metabolites. PSCs were cultured for 48 h in 20% Gln and 1 mM D-glucose with or without 10 mM Na-lactate. n = 3 biologically independent samples. (I) [U-13C]-Lac tracing in PSCs cultured in 100% or 20% Gln for 48 h. n = 3 biologically independent samples. (J) [1-13C]-Lac tracing. PSCs were cultured for 48 h in 100% or 20% Gln in the presence of 10 or 1 mM D-glucose and 10 mM Na-lactate. n = 3 biologically independent samples. (K) Western Blot of PSCs cultured in 20% Gln and the indicated concentrations of D-glucose and Na-lactate for 48 h. (L) Western Blot of PSCs expressing Ctrl or PC sgRNA, cultured in 20% Gln and the indicated concentrations of D-glucose and Na-lactate for 48 h. Mean+SD (A-F,H-J) are shown. Two-sided unpaired t-test (B), one-way ANOVA (C,D,J), one-way ANOVA with Holm-Sidak correction (E,F), two-sided unpaired t-test with Holm-Sidak correction (I). Western blots (G,K,L) are representative of two independent experiments. [U-13C]-Lac tracing in PSC in low glucose (H) was performed once. All other experiments were performed at least twice.
