Extended Data Fig. 5: Additional details of Golgi stress, altered glycosylation, and signaling/cell cycle changes following UXS1 ablation.
From: Disruption of sugar nucleotide clearance is a therapeutic vulnerability of cancer cells
(a) ARF4 immunoblots in UGDH high (UXS1 (A549) iKO and H1944) and low (HT1080) cells, 7 days post UXS1 KO, showing that ARF4 is only induced only in UGDH high cells following UXS1 ablation. (b) Changes in Oligomannose glycans (c) Paucimannose glycans (d) Sialylated glycans and (e) glycans with poly-LacNac extensions in A549 cells subjected to CTRL or UXS1 KO (n = 2 per group). (f) O-glycan profiling analysis performed on A549 cells subjected to CTRL or UXS1 KO at 7d post-transduction (n = 2 per group). (g) Immunoblots of phospho-histone H3 and actin from A549 UXS1 iKO and control iKO cells at indicated timepoints post doxycycline induction. Nocodazole-treated cells are used as a positive control for enrichment for cells in M phase. (h) Dot plot of total Golgi area per cell, from A549 UXS1 iKO cells induced via doxycycline, then labeled using GM130 at indicated days post-induction. No serum (orange) cells were serum starved for 12 h before labeling, which results in G0 cell cycle arrest and synchronization. p values from left to right are p = 0.48, p = 0.21, p = 0.15, p = 0.044. (i) GSEA enrichment plot showing that EGFR signaling and Cell Cycle genesets are highly depleted in UXS1 KO A549 cells compared to CTRL KO (n = 50 cells per group). (j) Same findings in another UXS1 sensitive cell line (H460). Cell Cycle genes are the most depleted geneset for ‘C2; canonical pathways’ (as shown in Fig. 3a), while EGFR Signaling genes are the most depleted geneset for the ‘C2; chemical and genetic perturbations’ ontology scheme. Data are shown as mean ± s.d. except in h where box plot shows median (centre) with interquartile range of 25% to 75%, minima and maxima. h, Two-tailed unpaired Student’s t-test. (n.s.: not significant, *p < 0.05).
