Fig. 4

NEU1 suppresses cell proliferation and induces apoptosis by inhibiting the Akt pathway. a Lectin blotting assay: Total proteins from YTS-1/Ctrl and YTS-1/NEU1 were extracted and subjected to SDS-PAGE. Biotin-labeled lectin blotting was performed, and signals were detected using a Vectastain ABC kit (Vector Laboratories; Burlingame, CA, USA). Coomassie blue staining (CB) of SDS-PAGE was used as loading control. b Cell morphology assay: YTS-1/Ctrl and YTS-1/NEU1 cells were seeded on 6-cm plates with complete culture medium, washed with ice-cold PBS, cultured 24 h, fixed with 4% fresh polyformaldehyde, and stained by crystal violet. Scale bar: 50 μm. c Cell proliferation (MTT assay). YTS-1/Ctrl, YTS-1/NEU1, T24/Ctrl, and T24/NEU1 cells (4 × 10³/well) were seeded on 96-well plates, and cultured for 6, 24, 36, 48, 60, or 72 h. Proliferation was assessed by MTT assay, and data were analyzed by the Prism software program and presented as mean ± SD from triplicate experiments. *, P = 0.01–0.05. **, P = 0.001–0.005. ***, P < 0.001. d Cell cycle analysis of YTS-1/Ctrl and YTS-1/NEU1. Effect of NEU1 over expression on cell cycle in YTS-1 cells, as measured by PI staining and flow cytometry. e The quantification of cell population distribution in different stages of cell cycle. f Cell apoptosis (FACS analysis). Cells were cultured overnight, detached by trypsin, incubated with Annexin V-FITC and PI for 20 min in the dark, and subjected to flow cytometry. Data were analyzed using the FlowJo software program. Representative results from two independent experiments are shown. g Cell cycle and apoptosis markers detected by Western blotting. Cyclin D1 and CDK2 were used as cell cycle marker. Bcl-2 and Caspase9 were used as apoptosis marker. h Total proteins from the four cell lines were extracted and subjected to SDS-PAGE and Western blotting. Signals for NEU1, FN, α5, β1, Akt, and phosphorylated Akt were revealed using Supersignal Chemiluminescence substrate kit as described in Supplementary information / Methods. i YTS-1 cells were cultured in complete medium for 12 h, and incubated with sialidase NEUA3 in serum-free medium for 30 min. Expression of FN, α5, β1, Akt, and phosphorylated Akt was analyzed by Western blotting. j HCV29 cells were transiently transfected with scrambled siRNA (control) or with two NEU1-specific siRNAs. Expression of NEU1, FN, α5, β1, Akt, and phosphorylated Akt was analyzed by Western blotting. Tubulin: loading control. Experiments were performed in triplicate, and representative blots are shown (a, g, h, i, j)