Extended Data Fig. 4: PC is required for collagen synthesis when extracellular glutamine is low.
From: Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment
(A) Western Blot of NIH-3T3 cells expressing Ctrl or PC sgRNA, cultured in 100% Gln for 48 h. (B) Western Blot of PSCs expressing Ctrl or PC sgRNA, cultured in 20% Gln for 48 h. (C) Western Blot of parental MFBs and MFBs expressing Ctrl or PC sgRNA, cultured in 100% or 20% Gln for 48 h. (D) Collagen abundance in ECM derived from confluent PSCs (left) or MFBs (right) expressing Ctrl or PC sgRNA, cultured in 20% Gln. n = 3 biologically independent samples. (E) Western Blot of MFBs expressing Ctrl or PC sgRNA, cultured in 20% Gln and treated with dm-αKG for 48 h. (F) [3,4-13C]-Glc tracing in NIH-3T3 cells expressing Ctrl or PC sgRNA cultured in 100% or 10% Gln for 48 h. n = 3 biologically independent samples. (G-I) Growth curves of NIH-3T3 cells (G), PSCs (H) or MFBs (I) expressing Ctrl or PC sgRNA, cultured in 100% or 10%/20% Gln. n = 3 biologically independent samples. (J) tRNA charging in PSCs (left) or MFBs (right) expressing Ctrl or PC sgRNA, cultured in 20% Gln for 48 h. n = 1 independent experiment. (K) Col1a1 mRNA expression in PSCs (left) or MFBs (right) expressing Ctrl or PC sgRNA, cultured in 20% Gln for 48 h. n = 3 biologically independent samples. (L,M) H3K27ac (L) or H3K27me3 enrichment (M) in NIH-3T3 cells expressing Ctrl or PC sgRNA, cultured in 10% Gln for 48 h. n = 3 (L), n = 4 (M) independent experiments. (N,O) Col1a1 mRNA expression in NIH-3T3 cells expressing Ctrl or PC sgRNA, cultured in 10% Gln for 48 h. Cells were treated with dm-Glu (N) or dm-αKG and MSO (O). n = 3 biologically independent samples. Mean SD (G-I), mean+SD (D, F, K-O) are shown. Dashed lines (G-I) represent cell number at day 0. Two-way ANOVA (F-I), one-way ANOVA (D,K), two-way ANOVA (L,M) analyzing the effects of PC-ko on H3K27ac or H3K27me3 across the analyzed genomic regions, one-way ANOVA with Holm-Sidak correction (N,O). Western blots are representative of two (A,E) or three (B,C) independent experiments. tRNA charging analysis (J) is representative of two independent experiments. All other experiments were performed at least twice.
