Extended Data Fig. 3: Deletion of GPI leads to metabolic reprogramming.
a) Proliferation of control (sgControl) and GPI knockout Akt1myr;MycOE;Trp53–/– cells (sgGPI #1/#4 and sgGPI #4 cl.1) cultured under hypoxia (0.5% O2). Data are displayed as mean ± SD (0 h n = 4, all others n = 8 biologically independent replicates). b) Western blot probing for GPI and Vinculin after 48hrs doxycycline (1ug/mL) treatment of shRen, shGPI.213, shGPI.1604 and shGPI.1544 expressing Akt1myr;MycOE;Trp53-/- cells. c) Proliferation of shRen, shGPI.213, shGPI.1604 and shGPI.1544 expressing Akt1myr;MycOE;Trp53–/– cells treated with doxycycline (1 µg/mL) or EtOH (solvent). Confluency was detected using live cell imaging of parallel wells. Data are presented as mean values ± SD (shRen and shGPI.155 EtOH n = 11, all others n = 12 parallel wells). d) Deletion of Gpi in human liver cancer cells (HLF, HLE and SNU-387) using CRISPR/Cas9. e) Proliferation of human liver cancer cells after depletion of GPI (sgGPI #1/#3) or control (sgCtrl). Cell mass was detected by crystal violet staining of independently seeded wells. Data are presented as mean values ± SD (HLE: n = 9; SNU387 sgGPI #1/#3 92 h n = 2, all others n = 3; HLF n = 8). f) Glycolytic activity of control (sgControl) and GPI knockout cells (sgGPI #1/#3) Cells were analysed using the glycolytic stress test (Seahorse). Data are presented as mean values ± SD (n = 15 parallel wells). g) Glycolytic activity of shRen, shGPI.213, shGPI.1604 and shGPI.1544 expressing Akt1myr;MycOE;Trp53–/– cells. Cells were analysed using the glycolytic stress test (Seahorse). Data are presented as mean values ± SD. Significance was calculated using one-way ANOVA with Dunnett’s post-hoc test (shRen and shGPI.1604 n = 21, all others n = 23 parallel wells). h) Diagram indicating the flux of 13C-labelled carbon atoms from 1,2-13C-Glucose into glycolysis and the pentose phosphate pathway (PPP). i) Isotopologue distribution at steady state for 3-phophoglycerate (3PG) in control cells (sgControl) and Gpi deleted cells (sgGpi #1/#4 or sgGPI #4 cl. 1). Table shows the estimated isotopologue distribution for 3PG assuming 100% flux through glycolysis or 100% flux through PPP69 in addition to the observed experimental results. Data are presented as mean values ± SD (n = 2 biologically independent replicates). j) Non-linear fit of relative enrichment of 13C labelled 3PG after 5, 10, 30 and 240 min of culture in 1,2-13C-Glucose containing medium, (E) is maximum enrichment at isotopic steady state and (f) is calculated flux. Data are displayed as mean ± SD (n = 2 biologically independent replicates).
