Fig. S1. Gonococcal protein is important for ATF3 induction. MS11 was incubated at 37°C (Live) or 50°C (HK) for 30 min and then treated with 3 or 6 units of proteinase K (PK) for 1 h. After washing out residual PK, T84 cells were infected for 4 h. Representative Western blot shows ATF3 levels after PK treatment. Total β-tubulin serves as a loading control.

Fig. S2. Tfp are not required to induce ATF3 expression.

A. Representative Western blot of ATF3 protein levels in T84 cells treated with increasing amounts of OMP isolated from MS11ΔpilE. β-Tubulin was used as a loading control.

B. Representative Western blot of ATF3 levels in cells infected with ΔpilE bacteria that were incubated at 37°C, 50°C or 100°C for 30 min prior to infection.

C. Densitometry values from (B) represent mean ATF3 protein levels, normalized to β-tubulin and set relative to mock-infected samples, which are set at 1 (± SEM, n = 4).

Fig. S3. PKR is phosphorylated by N. gonorrhoeae infection, but not required for ATF3 upregulation.

A. Representative Western blot showing treatment with a chemical PKR inhibitor blocks gonococcal-induced ATF3 upregulation. T84 cells were treated with vehicle DMSO or 5 μM of PKR inhibitor 52740 (PKRi) for 1 h and then infected with MS11 for 4 h. Cell lysates were harvested at 3 h post infection and ATF3 levels determined by Western blot.

B. Representative Western blot of PKR knock-down by RNA interference. T84 cells were nucleofected with nonsense (NS) or PKR-specific (PKR) siRNA. At 72 h post nucleofection, the cells were infected with MS11 for 3 h and protein levels determined by Western blot.

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