Pyruvate carboxylase promotes glycolysis and progression of endometriosis by activating the AKT pathway

Quasi-targeted metabolomics was employed to investigate the effects of PC knockdown (si#1) on cellular metabolite profiles, with experimental analysis performed by Novogene. The workflow consisted of four main phases: sample collection, metabolite extraction, mass spectrometry analysis, and bioinformatics processing. IhESCs in NC or siPC group were digested and counted. A total of 5×10⁵ cells were transferred into a centrifuge tube, centrifuged, and washed once with pre-chilled PBS. After another round of centrifugation, 300μL of 80% aqueous methanol solution was added, followed by quick-freezing in liquid nitrogen for 5 minutes. The mixture was thawed on ice, vortexed for 30s, and sonicated for 6 minutes. Subsequently, it was centrifuged at 5000rpm and 4℃ for 1 minute. The supernatant was collected into a new centrifuge tube, lyophilized to a dry powder, and then reconstituted in 300μL of 10% aqueous methanol solution. Metabolite detection was conducted using the SCIEX QTRAP® 6500+ mass spectrometer in multiple reaction monitoring mode, supported by Novogene's proprietary metabolomics database (novoDB). Quantification was based on Q3 (product ion) signals, while qualitative analysis integrated three-dimensional identification: 1) retention time alignment, 2) Q1/Q3 (precursor/product ion) pair matching, and 3) verification through MS/MS spectral library comparison. Three biological replicates were performed, and quality control (QC) samples were prepared by equal-volume mixing of all experimental samples. Data normalization was conducted using the total peak area normalization method. R software and MetaboAnalyst were utilized for bioinformatics analysis of metabolomics data, including principal component analysis (PCA), differential metabolite analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.