Fig. 3.

Infection of active, but not inactive AIEC LF82, leads to the increase of UDP-GlcNAc in HCT116 and HT-29 cells. (a - c) HCT116 cells were infected with AIEC strain LF82 (a), HB101 (b), and DH5α (c) at MOI of 10 for 2 h. Heat-inactivated respective bacteria were used in parallel. After the infections, cells were washed and cell lysates were used for the UDP-GlcNAc measurement. Levels of UDP-GlcNAc in mock-treated cells were set as 100%. The percentages of UDP-GlcNAc in infected cells to mock-treated cells were displayed. Values represent the mean ± S.D. ** p < .01 as compared with mock-treated and inactivated bacteria-treated groups, n = 3 (Student's t-test). (d - f) The levels of UDP-GlcNAc were determined in HT-29 cells with treatments and measurement as describled in a-c. (g,h) HCT116 cells were pre-treated with 10 μM 2-phenylbenzofuran for 1 h before infection with AIEC strain LF82 (MOI = 10) for 2 h. After the infection, cells were washed and cell lysates were collected. (g) Meaurement of UDP-GlcNAc in the cell lysates. Level of UDP-GlcNAc in mock-treated cells was set as 100%. The percentages of UDP-GlcNAc in AIEC LF82-treated cells with or without 2-phenylbenzofuran to mock-treated cells were displayed. Values represent the mean ± S.D. ** p < .01 as compared with infection cells without 2-phenylbenzofuran treatment, n = 3 (Student's t-test). (h) Detection of O-GlcNAc in the cell lysates by western blot. Thirty micrograms of whole cell extracts were loaded for each lane. GAPDH was detected for loading control. (i,j) UDP-GlcNAc and O-GlcNAc were determined in HT-29 cells with treatments and measurement as describled in g,h.