Figure 5.
Knockdown of Sirt4 prevents redox imbalance in the presence of Lsd1 inhibitor. (a–g) Comparison of TSCs cultured in the presence of Lsd1 inhibitor 1 (Lsd1i-1) or solvent and transfected with an unrelated or two different siRNAs directed against Sirt4. (a) Western blot decorated with the indicated antibodies. Band intensity was normalized to β-actin relative to the Ctrl transfected with an unrelated siRNA in the absence of Lsd1 inhibitor. (b) 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. (c) Relative mitochondrial membrane potential was determined by FL2/FL1 ratio. The background level was assigned by depolarization with CCCP. (d) Relative quantification of ROS using fluorescent dye. (e–g) Relative ratio of oxidized to reduced NADP and NAD (e), glutathione levels (f) and glutamine uptake (g). Data were analyzed from at least three biological samples and are represented as mean+S.E.M. *P<0.05, ***P<0.001 (unpaired, two-tailed Student's t-test, (b–g))