Figure 1.

Differential fluorescent labeling of intracellular B. thailandensis upon siRNA knockdown of selected host kinase genes. (A) THP-1 cells pretreated with 50 nM siRNA for 72 h were infected with FITC-labeled Bt CDC2721121 for 2 h at MOI 10. Kanamycin (250 μg/ml) was then added for 3 h to suppress extracellular bacterial growth, after which the infected cells were cultured in antibiotic-free media for 20 h. Bacterial cells remaining extracellular were double-labeled with FITC and B. thailandensis rabbit antiserum/PE-conjugated anti-rabbit IgG, whereas intracellular bacteria were single-labeled with only FITC. Imaging flow cytometry analysis of infected THP-1 cells was performed using the Amnis ImageStream^X, and the frequencies of singly and double labeled bacteria were determined using the IDEAS 5.0 software. FITC, fluorescein isothiocyanate (ex495 nm/em519 nm); PE, Phycoerythrin (ex 488 nm/em575 nm). (B) The ratio of extracellular vs. intracellular bacteria in THP-1 cells siRNA-depleted of target kinases was used to evaluate kinase function in intracellular B. thailandensis proliferation. The average and standard deviation were determined from three independent experiments (Supplementary Table 2). The “^*” denotes statistical significance (p < 0.05) for the ratio of extracellular to intracellular pathogen in cells siRNA-depleted of target gene compared to cells treated with control siRNA.