FIG 4.

GBS-mediated NF-κB signaling in macrophages occurs downstream of MAPK activation. THP-1 macrophages were treated with a p38 inhibitor (SB203580, 10 μM), a JNK inhibitor (SP600125, 25 μM), or a vehicle control (DMSO) 1.25 h prior to infecting them with GBS at a MOI of 10 for 1 h. The cells were washed and treated with antibiotics for an other hour prior to fixation, nuclear staining (DAPI), and detection of NF-κB p65 (Alexa Fluor 488) by immunofluorescence microscopy. The percentage of NF-κB nuclear localization compares the number of cells with positive nuclear localization (Alexa Fluor 488) to the total cell number in a given field (DAPI) using ImageJ. Results from three independent biological replicates were pooled for each condition, each of which was obtained from at least three separate fields for a minimum of 2,200 cells per condition. (A) ANOVA (P < 0.0001) and a post hoc Tukey’s test (*, P = 0.01 to 0.05; **, P = 0.001 to 0.01; ***, P = 0.0001 to 0.001; ****, P < 0.0001) were used to compare the mean of each ST or CPS group to the mean of every other ST or CPS group. For simplicity, only statistical differences for the GB411 infection conditions are shown. There was no statistical difference between the mock infection vehicle control and mock infection inhibitor treatments, but GB411 conditions were all significantly increased compared to all mock infection conditions. (B) Representative microscopy images of NF-κB localization (Alexa Fluor 488) for each condition are shown.