Fig 2: Knockdown of ST6GAL1 expression correlates with increased resistance to vincristine. Paired sets of control BCP-ALLs and cells with ST6Gal1 knockdown were plated on mitotically inactivated OP9 cells and treated for 15 days with the indicated concentrations of vincristine. (A, D) Values mean ±SEM of n = 3–4 replicates per time point per sample. (B, C) Four to six replicates per time point per sample. Two-way ANOVA, Šidák’s multiple comparison test, adjusted p-values, *p < 0.05, **p < 0.01; ***p < 0.001, ****p < 0.0001.

Fig 3: Overexpression of ST6Gal-I in Suit2 cells promotes activation of metastasis-associated gene networks including stemness and epithelial to mesenchymal transition (EMT) pathways. A, graph depicts the top 20 IPA Biological Functions altered in Suit2 overexpression (OE) cells as compared with Suit2 empty vector (EV) cells. Fourteen of the Biological Functions altered in Suit2 OE cells were also altered in both S2-LM7AA and S2-013 cells (red bars). Another two Functions were shared with one of the two subclones (green bars). B, gene Set Enrichment Analysis of Suit2 OE versus EV cells show that OE cells are enriched in the following pathways: Wnt (normalized enrichment score [NES] = 1.34; false discovery rate [FDR] = 0.10), Hedgehog (NES = 1.84, FDR = 0.001), EMT (NES = 2.24, FDR < 0.0005) and Hypoxia (NES = 1.85, FDR = 0.001).

Fig 4: Extracellular ST6GAL1 compensates for cell-intrinsic ST6GAL1 actions in breast tumor cells.A 4T1.2 cells transfected with shControl or mouse shST6GAL1 #1 construct and invasion abilities were assessed with 3D spheroid cell culture setting after 48 h of treatments, as indicated. Representative images and quantification of cancer cell invasions are shown (A, upper and lower panel, respectively). Scale bar 250 µm, N = 5 spheroids, data are means ± s.e., ANOVA, post-hoc t-test, p < 0.001 vs. respective controls. B BT-549 cells transfected with shControl, human shST6GAL1 #1 or shST6GAL1 #2 were treated with the control medium, self-exosome particles, rST6G, or self-exosomes, rST6G in combination, and 3D invasion assays were performed. The scale bar 250 µm (B, upper panel) shows representative phase-contrast microscopy images. Quantification of invasions (B, lower panel), n = 5, data means ± s.e, ANOVA, post-hoc test, p < 0.0001 vs. respective controls. Separate cultures were used to validate knockdown efficiencies of ST6GAL1 in 4T1.2 and BT-549 cells by qPCR and Western blotting, Supplemental Fig. S5 panel A, B, respectively.

Fig 5: ST6GAL1 knockdown impedes cellular reprogramming and establishment of induced pluripotency in human somatic cells.(a) Schematic illustration of the experimental strategy to examine the influence of ST6GAL1 knockdown on cellular reprogramming. Twenty-four hours after HDFs received POU5F1, SOX2, KLF4, and MYC with or without ST6GAL1 shRNA, the transduced HDFs were seeded on X-ray irradiated feeder cells (mouse embryonic fibroblasts, MEFs). Fourteen days later, alkaline phosphatase (AP) staining was used to examine hiPSC colonies formed by transduced HDFs on the feeder cells. (b) AP staining showed that dramatically fewer hiPSC colonies with AP activity were obtained from cellular reprogramming under ST6GAL1 knockdown mediated by shRNA2 and shRNA5. (c) Quantitative analysis of NANOG expressing cells in the reprogrammed cell population showed that shRNA2 and shRNA5 both led to a significant reduction in NANOG expressing cells in the analyzed cell populations. Left Panel: The histogram representation of flow cytometry analysis. Right panel: the quantitative result of flow cytometry analysis (n = 3; *P < 0.05, t-test). (d) The expression of endogenous POU5F1, SOX2, KLF4 and MYC genes in cells that underwent reprogramming with or without ST6GAL1 knockdown was measured by qRT-PCR with primer sets that target untranslated regions of the endogenous gene transcripts. The induction of endogenous POU5F1 and SOX2 gene expression at the early stage of cellular reprogramming in HDFs was substantially suppressed by shRNA-mediated ST6GAL1 knockdown (n = 3; *P < 0.05, t-test). Day 6: cell samples collected at 6 days after the initial transduction (2 days after the beginning of puromycin selection). Day 12: cell samples collected at 12 days after the initial transduction (8 days after the beginning of puromycin selection).