File S1 - Pseudomonas aeruginosa Cytotoxicity Is Attenuated at High Cell Density and Associated with the Accumulation of Phenylacetic Acid

Figure S1. The 1H NMR spectrum of PAA. Figure S2. The 12C NMR spectrum of PAA. Figure S3. HPLC profile and UV spectrometry analysis of synthetic PAA. (A) HPLC of synthetic PAA, (B) UV spectrum of synthetic PAA (C) HPLC of purified PAA. (D) UV spectrum of purified PAA. Figure S4. Comparative analysis of synthetic and purified PAA. (A) Different dosages of synthetic PAA were used to protect A549 against PAO1 infection, and compared with purified PAA. (B) The effect of different dosages of synthetic PAA on growth of A549. Viability was measured by WST-1 test. Data are expressed as the percentage of internal control for each cell type measured 12 h after cell seeding (time 0) and are the mean ± SEM of 3 replicates. The experiment was repeated at least three times. Figure S5. Protection effect of different dosages of PAA and IAA against PAO1 infection. Control is normal A549 without bacterial infection. Viability was measured by WST-1 test. The experiment was repeated three times and the data shown are the means of 3 replicates with standard deviation and expressed as the percentage of control. Figure S6. PAA affects PAO1 virulence by down regulation of T3SS, not by inhibition of bacterial growth. (A) Effect of PAA on T3SS gene expression. The exsCEBA Promoter- directed β-galactosidase activity was determined 4 h after culture of the bacterial cells with different dosages of PAA. (B) Effect of PAA on bacterial growth. OD600 of bacterial cells (PAO1) was measured 4 h after culture of the bacterial cells with different dosages of PAA. Table S1. Strains and plasmids used in this study. Table S2. Primers for real time PCR. Table S3. 1H and 13C NMR of PAA in Methanol-d4. Table S4. Down-regulated genes in strain PAO1 after treatment with PAA. Table S5. Up-regulated genes in strain PAO1 after treatment with PAA. Table S6. T3SS regulator change after PAA treatment. (PDF)