Bafilomycin a1 bafa1

AGS cells (3 × 10⁵) were seeded in six-well plates and cultured for 16 h. The cells were treated with rCdtC (200 and 500 nM) or 0.1 μM of bafilomycin A1 (BafA1) (InvivoGen, San Diego, CA, United States) for 1 h prior to H. pylori infection at a synchronized MOI of 100 for 6 h. The elongated cells (hummingbird phenotype) were defined as cells that showed thin needle-like protrusions of more than 20 μm in length and a typical elongated shape, as reported previously (Wang et al., 2012 (link)). The percentage of elongated cells was determined as the number of cells having the hummingbird phenotype.

Dendritic cell maturation was induced by simultaneous addition of 100 ng ml⁻¹ LPS (ultrapure lipopolysaccharide from Salmonella minnesota R595, Cat. No. tlrl-smlps) and 10 ng ml⁻¹ recombinant human IFNγ (PeproTech, Cat. No. 300-02) for the indicated time periods. For autophagy induction, moDCs were exposed to 50 nM rapamycin (Merck, Cat. No. 553210) for 4 h. The optimal concentration of rapamycin was determined as one that readily increased LC3-II levels without significant toxicity. In some experiments, cells were incubated with 20 nM bafilomycin A1 (BafA1) (InvivoGen, Cat. No. tlrl-baf1) or 1 µM MG132 (SelleckChem, Cat. No. S2619) for the last 2 h.

Cells in exponential growth phase were inoculated at a concentration of 2.0 × 10⁶ cells/mL into 125-mL Erlenmeyer flasks, each containing 30 mL of SFM4CHO with 4 mM glutamine.
The autophagy inhibitors, SP600125, LY294002, and bafilomycin A1 (BafA1) (InvivoGen, San Diego, CA, USA) were prepared by dissolving them in dimethyl sulfoxide (DMSO) and were individually added to the culture at a concentration of 30 μM, 20 μM, and 5 nM, respectively, on day 0. Cell culture without any chemical addition was used as a control, and the same volume of solvent (DMSO) was used as a vehicle control.
The nucleotide sugar precursors, N-acetylmannosamine (ManNAc), galactose (Gal), glucose (Glc), glucosamine (GlcN), and N-acetylglucosamine (GlcNAc) (Sigma-Aldrich, St. Louis, MO, USA), were added to the cultures at concentrations of 20, 10, 10, 10, and 10 mM, respectively, on day 0. Specifically, ManNAc, Gal, and GlcNAc were combined as a mixture of nucleotide sugar precursors, and this mixture was treated with autophagy inhibitors on day 0.
Cell culture samples were taken daily from the flasks to determine the viable cell density, viability, and Fc-fusion glycoprotein concentration and were kept at −70 °C for further analysis.