Figure 3

Lsd1 regulates anaplerosis and maintains redox balance. (a–m) Comparison of TSCs cultured for 24 h in the presence of solvent (Ctrl), Lsd1 inhibitor 1 (Lsd1^i-1) (a–m), and Lsd1 inhibitor 2 (Lsd1^i-2) (b–m). (a) Metabolomics profile depicted by log₂ fold change versus −log₁₀ P-value. Schema and bar graph depicting increased (red) and decreased (blue) metabolites of glycolysis, TCA cycle and glutamine anaplerosis. (b–e) Quantification of glycolysis rate by extracellular acidification rate (ECAR) (b), relative glutamine uptake (c), Glud1 activity (d) and ammonia levels (e). (f) Mitochondrial respiration was determined by a time course of OCR. Complex V was blocked by oligomycin (O), uncoupling was induced by FCCP (F) and electron transport system was disabled by addition of rotenone (R) and antimycin A (A). Basal respiration is the difference of the OCR in the absence of inhibitors and after addition of rotenone and antimycin A. The reserve capacity is derived from the subtraction of the OCR after addition of rotenone and antimycin A from oligomycin-treated TSCs. (g) Relative mitochondrial membrane potential determined by FL2/FL1 ratio. The background level was assigned by depolarization with CCCP. (h–m) Measurement of ATP concentration (h), relative H₂O₂ concentration determined with cytoplasmic and mitochondrial ratiometric reporters (i), relative quantification of ROS using fluorescent dye (j), relative concentration of oxidized glutathione (GSSG) determined by cytoplasmic and mitochondrial ratiometric reporters (k), relative ratio of oxidized to reduced NADP and NAD (l) and relative glutathione levels (m). Data were analyzed from at least three biological samples and are represented as mean+S.E.M. *P<0.05, **P<0.01, ***P<0.001 (unpaired, two-tailed Student's t-test)