Fig. 4
Knockdown of MTHFD2 and oxPAPC drains the intracellular glycine pool. a Heatmap for fragments per kilobase of transcript per million mapped reads (FPKM) of significantly differentially expressed genes (FDR < 0.01, Benjamini−Hochberg). RNAseq was performed in HAEC with three different siRNAs against MTHFD2 or scrambled control. b Projection of RNAseq signature (FDR < 0.01, Benjamini−Hochberg) in a onto the MTHFD2 network. Direction of expression of nodes of the MTHFD2 network in RNAseq signature is indicated by the node color. Node size reflects out-degree. c Venn diagram of genes in the amino acid cluster, MTHFD2 network and MTHFD2 RNAseq signature (FDR < 0.01, Benjamini−Hochberg). Genes belonging to all three gene sets are listed. d Heatmap of amino acid profile. HAEC were treated with three different siRNAs against MTHFD2 or scrambled control and exposed to medium (1% FCS) with or without oxPAPC for 4 h. Amino acids in cell lysates were measured by mass spectrometry (n = 6–9). e Relative mRNA expression of MTHFD2 in HAEC pretreated with N-acetylcysteine (NAC) (5 mM) and glycine (500 µM) for 1 h and then exposed to medium (1% FCS) with (oxP) or without (Ct) oxPAPC or tunicamycin (10 µg ml−1) for 4 h (n = 5). f qRT-PCR detection of MTHFD2 in HAEC exposed to medium (1% FCS) with or without oxPAPC and treated with tBHQ (20 µM) or DMSO as control for 24 h (n = 4). g HAEC were exposed to medium (1% FCS) with or without oxPAPC and additionally treated with torin (100 nM), ML385 (10 µM) or DMSO as control (n = 4). h, i Western blot detection (h) and quantification (i) of phosphorylated S6, S6 and MTHFD2 in HAEC pretreated with rapamycin (20 nM) or DMSO as control overnight and exposed to medium (1% FCS) with or without oxPAPC for 4 h (n = 4). j HAEC were treated with scrambled control siRNA (siCtr) or siRNA against ATF4 and exposed to oxPAPC or control medium for 4 h (n = 5). Data are represented as mean ± SEM, *p ≤ 0.05 (oxP or tunicamycin vs Ct), #p ≤ 0.05 (inhibitor present vs absent or ATF4 vs Control siRNA) (ANOVA with Bonferroni post-hoc test)
