FIG 4.

Effects of sialidase inhibitors (SB or NADNA) on sialidase activity and ETX production. (A and B) Inhibition of wild-type CN3718 TH culture supernatant sialidase activity by sialidase inhibitors was tested. (A) Experimental protocol. (B) Sialidase activities of wild-type CN3718 supernatant (Mock), as well as the same supernatant supplemented with 5× IC₅₀ of SB or NADNA. All experiments were repeated three times, and mean values are shown. The error bars indicate standard deviations. (C and D) Inhibition of CN3718 TH culture sialidase activity when the culture medium was supplied with sialidase inhibitors was tested. (C) Experimental procedure. (D) Sialidase activities of washed wild-type CN3718 cells cultured in TH medium, which were supplemented with 5× IC₅₀ of SB or NADNA for 4 h at 37°C. All experiments were repeated three times, and mean values are shown. The error bars indicate standard deviations. (E) Western blot of ETX production when TH medium was supplemented with SB (5× IC₅₀, 10× IC₅₀ or 20× IC₅₀). The protein size marker is shown to the right. The band intensities of the Western blot were compared by Image J analysis. All experiments were repeated three times; a representative result is shown. (F) Western blot of ETX production when TH medium was supplemented with NADNA (5× IC₅₀, 10× IC₅₀, or 20× IC₅₀). The protein size marker is shown to the right. The band intensities of the Western blot were compared by Image J analysis. All experiments were repeated three times; a representative graph of three independent experiments is shown.