Fig. 1

EDI3 silencing alters glycerophospholipid metabolism in ER-HER2+ breast cancer cells. A Schematic of the inducible lentiviral shRNA vector (SMARTvector™, Dharmacon) for doxycycline-dependent expression of shRNA oligos. B EDI3 mRNA expression and C representative Western blot showing EDI3 protein expression in HCC1954-luc shNEG and HCC1954-luc shEDI3 (oligos shEDI3 #1 and #2) cells after 72 h treatment with 0.01 or 0.1 µg/ml doxycycline. D Overview of the metabolic pathways downstream of EDI3. By hydrolyzing GPC to choline and G3P, EDI3 plays a role in choline metabolism via the Kennedy pathway as well as in the synthesis of signaling and structural glycerophospholipids. E Intracellular GPC/PCho ratio, and intracellular levels of F choline, G G3P, H 16:0 LPA and I 16:0 LPC measured using LC–MS/MS in HCC1954-luc shNEG and shEDI3 after 72 h treatment with 0.01 or 0.1 µg/ml doxycycline (left panel) as well as in SUM190PT (middle panel) and SKBR3 cells (right panel) after silencing EDI3 using two different siRNA oligos. Metabolite levels were determined by calculating the ratios of the integrated peaks of the endogenous metabolites and the internal standards. Quantities of metabolites were normalized to cell number and presented relative to untreated control cells (shNEG or siNEG). Data represent mean ± SD from at least five technical replicates (wells from a six-well plate) from one or two independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). GPC, glycerophosphocholine; PCho, phosphocholine; LPA, lysophosphatidic acid; PA, phosphatidic acid; LPC, lysophosphatidylcholine; LPS, lysophosphatidylserine; LPG, lysophosphatidylglycerol; DAG, diacylglycerol; PtdCho, phosphatidylcholine; CDP-Cho, cytidine diphosphate choline; PS, phosphatidylserine; G3P, glycerol-3-phosphate; CDP-DAG, cytidine diphosphate diacylglycerol; PGP, phosphatidylglycerol phosphate; PG, phosphatidylglycerol