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. 2021 Oct 15;89(11):e00256-21. doi: 10.1128/IAI.00256-21

FIG 8.

FIG 8

Reduction of mucus in MTX-E12 cultures using a NAC pretreatment to reduce adherent mucus affects F4969 growth, sialidase production, and adherence. (A) Muc5Ac Western blot analysis performed on an SDS-containing 8% polyacrylamide gel for MTX-E12 cells and MTX-E12 cells pretreated with NAC (see Materials and Methods). Size of proteins in kilodaltons is shown on the left. The same amount of each protein sample was loaded onto a SDS-containing 12% polyacrylamide gel or Western blotted with an anti-beta actin antibody for a loading control. Size of proteins in kilodaltons is shown at right. The gel results shown are representative of three repetitions. (B) Postinoculation changes in the OD600 for cultures of wild-type F4969 growing in PBS buffer with MTX-E12 or NAC-pretreated MTX-E12 cells at 37°C for 24 h. A 0.5-ml aliquot of each culture was removed, and the OD600 was determined. (C) Sialidase activity measured for F4969 growing in PBS buffer with MTX-E12 or NAC-pretreated MTX-E12 cells at 37°C for 24 h. Sialidase activity was measured in culture supernatants of samples from panel B. (D) Attachment of MTX-E12 or NAC-pretreated MTX-E12 cell monolayers incubated for 2 h with washed vegetative cells of F4969 at 37°C under anaerobic conditions. Monolayers were then washed three times with PBS. After collection, the adherent bacteria were plated onto BHI agar plates for counting. All panels show the mean values from three independent experiments. The error bars indicate the SD. *, P < 0.05 relative to coculture with MTX-E12 cell line.