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. 2019 Sep 24;10(5):e02144-19. doi: 10.1128/mBio.02144-19

FIG 1.

FIG 1

(A and B) RAW macrophages were incubated with FAM-SE-labeled bacteria preincubated in PBS, heat-inactivated (HK) serum, or 10% pooled human serum. Cells were washed at 2 time points and then lifted and analyzed by flow cytometry to assess association. The results of 4 experiments are displayed as means ± standard errors of the means (SEM) at (A) 15 min or (B) 60 min, with TIGR4 data shown as gray bars and TIGR4 Δcps data as white bars, as analyzed by t test. (C) MDMs incubated with bacteria at an MOI of 10 had an antibiotic protection assay performed after 2 h, and data were analyzed by t test, with means ± SEM from 3 experiments displayed. WT TIGR4 data are shown as black bars and TIGR4 Δcps data as gray bars. (D to F) High-throughput imaging of fluorescent TIGR4 and TIGR4 Δcps labeled with green fluorophore and incubated for 2 h with MDMs (nuclei in blue), with external bacteria labeled with anti-human IgG (labeled red) appearing yellow (red arrow) due to the interaction of red and green fluorophores, whereas internalized bacteria remain green (white arrow). Sample images of MDMs incubated with TIGR4 and TIGR4 Δcps are shown in panels E and F, respectively. (G) Numbers of external and internal bacteria were counted, and data are displayed as means ± SEM from 3 experiments, with analysis by unpaired t test. WT TIGR4 data are shown as black bars and TIGR4 Δcps data as gray bars. (G) Once TIGR4 and TIGR4 Δcps were internalized, with inocula adjusted such that internalized bacteria at time zero were similar, numbers of surviving internal bacteria were measured by antibiotic protection assay using THP-1 cells. Results are displayed as means ± SEM from 5 experiments at each time point, as analyzed by unpaired t test. WT TIGR4 data are displayed as squares joined by a solid line and TIGR4 Δcps data as circles joined by a dotted line. (H) Bacteria were incubated with MDMs at an MOI of 10, and MDM supernatant was plated at specified time points, displayed as the median and range from 6 experiments, with WT TIGR4 data shown as black bars and TIGR4 Δcps data as gray bars, with counts analyzed by the Mann-Whitney U test.