Extended Data Fig. 2: Effects of silencing and pharmacological inhibition of GS on EC viability and central metabolism.
From: Role of glutamine synthetase in angiogenesis beyond glutamine synthesis
a, GLUL mRNA levels in control ECs and ECs transduced with two different non-overlapping shRNAs targeting GLUL (GLULKD1 and GLULKD2; GLULKD1 is used in the experiments in the text and is denoted GLULKD) or transfected with scrambled siRNA (SCR) or siRNA targeting GLUL (siGLUL). Data are expressed as a percentage of the respective control, denoted by the horizontal dotted line (mean ± s.e.m.; n = 28 independent experiments for GLULKD1, n = 3 independent experiments for GLULKD2 and n = 9 independent experiments for siGLUL; *P < 0.05 versus the respective control; one-sample t-test). b, c, Quantification of number of sprouts (b) and total sprout length (c) for spheroid-sprouting assays with GLULKD ECs and GLULKD ECs expressing a shRNA-resistant GLUL mutant (rGLULOE) (mean ± s.e.m.; n = 3 independent experiments; *P < 0.05 and NS, P > 0.05 versus control; one-way ANOVA with Dunnett’s multiple comparison versus control). d, Viability of control (Ctrl) and GLULKD ECs as measured by lactate dehydrogenase (LDH) release assay (mean ± s.e.m.; n = 3 independent experiments; NS, P > 0.05 versus control, one-sample t-test). e, Intracellular levels of reactive oxygen species measured by CM-H2DCFDA staining (mean ± s.e.m.; n = 3 independent experiments; NS, P > 0.05 versus control, Student’s t-test). f, Energy charge measurement (([ATP] + 1/2[ADP]) / ([ATP] + [ADP] + [AMP])) in GLULKD and control ECs (mean ± s.e.m.; n = 3 independent experiments; NS, P > 0.05 versus control, Student’s t-test). g, Ratio of oxidized glutathione (GSSG) over total glutathione levels (GSSG/(GSH + GSSG)) in GLULKD and control ECs (mean ± s.e.m.; n = 4 independent experiments; NS, P > 0.05 versus control, Student’s t-test). h, NADP/NADPH ratio in GLULKD and control ECs (mean ± s.e.m.; n = 5 independent experiments; NS, P > 0.05 versus control, one-sample t-test). i–k, Effect of GLUL knockdown on major metabolic fluxes including glycolysis (i), glucose oxidation (j) and glutamine oxidation (k) (mean ± s.e.m.; n = 3 independent experiments for i, n = 5 for j and n = 4 for k; NS, P > 0.05 versus control, one-sample t-test). l, m, Oxygen consumption rate (OCR) in control, MSO-treated and GLULKD ECs in basal state and after injection of oligomycin, FCCP and antimycin A (l) (mean ± s.e.m.; n = 3 independent experiments), and calculation of OCRBAS, OCRATP and maximal respiration (m) (mean ± s.e.m.; n = 3 independent experiments). Exact P values: GLULKD1: <0.0001; GLULKD2: <0.0001; siGLUL: <0.0001 (a); control versus GLULKD: 0.0147; control versus GLULKD + rGLULOE: 0.9824 (b); control versus GLULKD: 0.0083; control versus GLULKD + rGLULOE: 0.6528 (c); 0.5717 (d); 0.8206 (e); 0.3715 (f); 0.4398 (g); 0.9291 (h); 0.4691 (i); 0.6643 (j); 0.6786 (k). CM-DCF, CM-H2DCFDA; OCRBAS, basal oxygen consumption rate; OCRATP, ATP-generating oxygen consumption rate; RFU, relative fluorescence units.
