The epithelial cells were infected with bacteria to a multiplicity of infection (MOI) of 100. The bacteria were not removed after addition to the epithelial cells. In some experiments Millicell 0.4 μm filters (Millipore) were used to prevent direct bacterial contact to the host epithelial cells. For infection with dead bacteria, a dense suspension of bacteria was heat-killed at 95°C for 10 min and diluted in cell culture medium to the desired density for infection. After 1, 2, 4, and 6 h of incubation RNA was isolated using the RNeasy Plus kit (Qiagen) according to manufacturer's instructions. The concentration of the RNA was determined using the NanoDrop 8000 (Thermo Scientific) UV-Vis Spectrophotometer. Total RNA was reverse transcribed to cDNA using SuperScript VILO Mastermix (Invitrogen). Quantitative PCR was performed using a LightCycler 480 (Roche) and the SYBRGreen I Master kit (Roche). The primers used are listed in Table 2. The thermal cycling conditions were: initial denaturation at 95°C for 10 min followed by amplification for 40 cycles with denaturation at 95°C for 10 s, annealing at 50°C for 20 s and extension at 72°C for 20 s. The melting curve analysis was as follows: 95°C for 5 s, 65°C for 1 min and then increasing to 95°C at 0.08°C/s. The expression was normalized against the housekeeping gene β-actin. The expression levels were calculated by the comparative CT method (ΔΔ CT method) expressed as the fold change compared to uninfected cells.