Extended Data Fig. 3: Supporting data that UDPGA is toxic.
From: Disruption of sugar nucleotide clearance is a therapeutic vulnerability of cancer cells
(a) Relative viability of DLD1 cells subjected to preemptive CTRL (gray) or UGDH (faucet; orange) KO, followed by CTRL or UXS1 (drain) KO. Values are relative to cells subjected to CTRL KO then CTRL KO ( = 1.0) (n = 3 per group). (b) Immunoblot of UXS1 and UGDH protein levels under the KO combinations shown in a. (c) GC-MS quantification of UDPGA in cell-lines subjected to CTRL (gray) or UXS1 KO (pink) at 8 days of transduction (n = 3 per group). (d) Relative viability of A549 UXS1 iKO cells grown in media containing increasing glucose concentrations. Values are relative to uninduced cells grown in media containing 25 mM glucose (=1.0) (n = 3 per condition). (e) GC-MS quantification of UDPGA (blue; left Y axis) and relative viability (red; right Y axis) of A549 cells treated with increasing concentrations of UDPGA, showing dose-dependent increase in UDPGA and accompanying decrease in viability (n = 3 per group). (f) Viability of H838 cells overexpressing either blank vector (gray) or UGDH (red) then subjected to CTRL or UXS1 KO. Values are relative to blank vector overexpressing, CTRL KO cells (=1.0) (n = 3 per group). (g) Immunoblot of UXS1 and UGDH protein levels under the OE/KO combinations shown in c; LE indicates low exposure, HE indicates high exposure. (h) Relative UDPGA produced [M + 6 UDPGA / (M + 6 UDP-glucose/ total UDP-glucose)] in control, UGDH KO, and UXS1 KO A549 cells (n = 3 per group). (i) Labelled UDP-glucose (M + 6) in control and UXS1 KO A549 cells (<1 h) (n = 3 per group). (j) Unlabelled UDP-glucose (M + 0) in control and UXS1 KO A549 cells (<1 h) (n = 3 per group). (k) Total UDP-glucose (M + 6 + M + 0) in control and UXS1 KO A549 cells (<1 h) (n = 3 per group). (l) Fractional labeling of UDP-glucose [M + 6 UDP-glucose/ total UDP-glucose] of control and UXS1 KO cells (<1 h) (n = 3 per group). Data are shown as mean ± s.d. a, c-f, i, Two-tailed unpaired Student’s t-test. P values from left to right in a: p = 0.002, p = 0.0001. p = 7.9E-06, c: p = 0.52, p = 0.006, p = 0.013, d: p = 0.0006, p = 0.015, e: (UDPGA) p = 0.016, p = 0.035, p = 0.0001, (viability) p = 0.0007, p = 0.0008, p = 3.14E-05 f: p = 0.001, p = 0.011, p = 0.001 (n.s.: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
