IL-8 synthesis in HUVECs expressing constitutively active MKK6(Glu).

(A) HUVECs were either left untreated or incubated with 100 ng/mL LPS for 8 hours. IL-8 synthesis was measured by flow cytometry as described in “Materials and methods.” Flow cytometry profiles of 1 representative experiment are shown. Open profiles represent isotype controls; shaded profiles, cells labeled for IL-8 expression. Bold letters indicate mean fluorescence intensities. A total of 10 000 cells of each sample were analyzed. (B) HUVECs were transfected in a 1:3 ratio with pGreenLantern expressing GFPS65T and plasmids expressing either empty expression vector KRSPA or KRSPA MKK6(Glu). Cells were left untreated or treated 36 hours after transfection with 10 ng/mL TcdB-10463 or 100 ng/mL TcdB-1470 for 8 hours. Successfully transfected cells were identified by expression of GFPS65T and analyzed for IL-8 synthesis by flow cytometry as described. Flow cytometry profiles of 1 representative experiment are shown. A total of 5000 transfected cells were assessed of each sample. (C) Mean fluorescence intensities ± SD in fold of unstimulated or vector-transfected control cells of at least 2 experiments as described in (A) and (B) are shown.