Fig 1: MGAT3 knockdown promotes hypoxia-induced EMT in MCF7 cells (A) MGAT3 expression in mock- and MGAT3-shRNA-transfected MCF7 cells. Cells were stably transduced with lentivirus carrying anti-MGAT3 shRNAs (MCF7/shMGAT3-1/2) or shNC (MCF7/mock), harvested, and lysed in T-PER Reagent. Western blotting was performed as described in M&M. (B) Cell proliferation. The two transfectants were cultured for 24, 36, 48, 60, and 72 h, and proliferation was assessed by MTS assay. (C) Cell migration. Migration assays of the two transfectants under normoxic and hypoxic conditions were performed as described in M&M, and relative migration rate was shown (E). *p < 0.05; **p < 0.01. (D) Expression of HIF-1α, MGAT3, AKT, p-AKT, E-cadherin, fibronectin, β-catenin, and tubulin in the two transfectants under normoxic and hypoxic conditions. Cells were cultured as described in Figure 1, harvested, and lysed in T-PER Reagent. Protein content was determined by BCA assay. Western blotting was performed as described in M&M.
Fig 2: Effects of bisecting GlcNAc on migration, proliferation, clonal formation, and EGFR/ERK signaling activation. (A) Expression of MGAT3 and levels of bisecting GlcNAc in control (Vector) and MGAT3-overexpression MDA-MB-231 cells (two transfectants, termed MGAT3-1 and 2). (B) Migratory ability of MGAT3 and vector transfectants assessed by wound assay. Transfectant MGAT3-1 was used for following functional assay analysis. Cell monolayers were scratched with pipette tip. Cells were washed with ice-cold PBS and photographed at 0 and 24 h (100 × magnification). *p < 0.05. Cell proliferation of MGAT3 and vector transfectants by EdU incorporation assay (C) and CCK8 assay (D). Cells were incubated with 40 μM Edu for 6 h, then fixed with paraformaldehyde, stained and subjected to flow cytometry analysis. ***p < 0.001. (E) Colony formation of MGAT3 and vector transfectants. Two thousand five hundred cells were cultured in 6-cm dishes for 1–2 weeks, fixed, stained with crystal violet solution, and photographed. Acetic acid was used to dissolve crystal violet, and OD 595 was determined. *p < 0.05. (F) Invasion assay of MGAT3 and vector transfectants. 2 × 104 cells were plated in upper chamber coated with Matrigel. After 24 h culture, cells migrated across the membrane were stained with 0.1% crystal violet, and photographed under microscope (magnification 100×). ***p < 0.001. (G) Expression of EGFR with bisecting GlcNAc by immunoprecipitation in MGAT3 and vector transfectants. MGAT3 and vector transfectants lysate were subjected to immunoprecipitation with antibody against EGFR, and immunoprecipitates were subjected to immunoblot analysis. (H) Activation of EGFR associated signaling pathway in MGAT3 and vector transfectants by western blot. (I) Luciferase activity of nude mice at week 8 following injection of MGAT3-overexpressing MDA-MB-231 and parental cells (each n = 6 mice). **p < 0.01. (J) Representative photographs and metastatic node numbers of lungs from nude mices injected with MGAT3-overexpressing MDA-MB-231 and parental cells. *p < 0.05. (K) Hematoxylin and eosin (H&E) staining of lungs from nude mices injected with MGAT3-overexpressing MDA-MB-231 and parental cells.
Fig 3: Variation of fine glycan structures detected by lectin microarray analysis (A) Variation of levels of glycans from MCF7 (upper) and MDA-MB-231 (lower) cells, detected by 37 lectins, is presented as a heatmap. Lectin microarray analysis was performed as described as M&M. Red: fluorescence signal activation. Green: signal inhibition. Black: missing data. (B) Altered glycan levels evaluated by lectin histochemistry. Four lectins (Con A, MAL-I, LCA, PHA-E) were applied, and lectin histochemistry was performed as described in M&M. Signals are shown from merge images of Cy3-conjugated lectins and DAPI staining of nuclei in MCF7 (left) and MDA-MB-231 (right) under normoxic and hypoxic conditions (60× magnification). (C) Expression in MCF7 and MDA-MB-231 cells of HIF-1α, MGAT3, and tubulin.
Fig 4: MGAT3 overexpression suppresses hypoxia-induced EMT in MCF7 cells (A) MGAT3 expression in mock- and MGAT3-transfected MCF7 cells. Cells were stably transduced with a GFP-marked lentivirus carrying mock gene or MGAT3 gene, harvested, and lysed in T-PER Reagent. Western blotting was performed as described in M&M using anti-MGAT3 and anti-GFP antibody. (B) Levels of bisecting GlcNAc structures in mock- and MGAT3-transfectants. Whole cell lysates of the two transfectants were subjected to PHA-E lectin blotting as described in M&M. (C) Proliferation of transfectant cells. The two transfectants were cultured for 24, 36, 48, 60, and 72 h, and proliferation was assessed by MTS assay. (E) Colony formation ability. The two transfectants (2500 cells each) were cultured in 6-cm dishes for 1–2 week, fixed, stained with crystal violet solution, and photographed. Acetic acid was added to dissolve crystal violet, and OD 595 was determined (D). *p < 0.05; ***p < 0.001. (F) Cell migration. Migration assays of the two transfectants under normoxic and hypoxic conditions were performed as described in M&M, and relative migration rate was shown (H). *p < 0.05. (G) Expression of HIF-1α, MGAT3, AKT, p-AKT, E-cadherin, fibronectin, and tubulin in the two transfectants under normoxic and hypoxic conditions. Cells were cultured as described in Figure 1, harvested, and lysed in T-PER Reagent. Protein content was determined by BCA assay. Western blotting was performed as described in M&M.
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