cell lines human lung tissue cell line a549 atcc cat  (ATCC)

Journal: Cell reports

Article Title: Within-host evolution of a transcriptional regulator contributes to the establishment of chronic Pseudomonas aeruginosa infection.

doi: 10.1016/j.celrep.2024.115214

Figure Lengend Snippet: Figure 4. GavR* regulates bacterial growth and virulence by directly binding to the aceE promoter (A) The predicted 26-bp GavR-binding sites identified by MEME analysis. By analyzing the results of ChIP, the target sequence was aligned to identify the 26-bp binding site of GavR. (B) The predicted 15-bp binding sites of GavR and GavR* identified by sequence analysis. (C) The transcription levels of aceE in strains PAO1, DgavR, and gavR* were represented by the FPKM value of aceE in the RNA-seq. Error bars represent standard deviation from the mean. ****p < 0.0001, two-tailed t test. (D) Growth curve assays assessed the effect of overexpression of aceEF compared to empty vector (EV) control in strain gavR* background. (E) The relative mRNA levels of the T3SS genes, including exsA, exoS, and pcrV, were determined by qPCR. Strains gavR* and gavR* with aceEF overexpression were grown in LB with 5 mM EGTA to an OD600 of 0.3. Error bars represent standard deviation from the mean. **p < 0.01, two-tailed t test. (F) Cytotoxicity of strains PAO1, gavR*, and gavR* with aceEF overexpression, DaceE, and the complementation strain of DaceE. A549 cells were infected with the indicated strains at an MOI of 100 for 7 h. The relative cytotoxicity was determined by the LDH release assay. Error bars represent standard deviation from the mean. *p < 0.05 and **p < 0.01, two-tailed t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GavR This study N/A Bacterial and virus strains E. coli DH5a Laboratory stock N/A E. coli BL21 Laboratory stock N/A P. aeruginosa PAO1 Laboratory stock N/A P. aeruginosa DgavR This study N/A P. aeruginosa gavR* This study N/A P. aeruginosa DaceE This study N/A P. aeruginosa DrpoS This study N/A P. aeruginosa gavR*DrpoS This study N/A P. aeruginosa F1498 Laboratory stock N/A P. aeruginosa F1498 DgavR This study N/A P. aeruginosa F1498+9bp This study N/A Biological samples Sputum samples This study N/A Chemicals, peptides, and recombinant proteins Disuccinimidyl suberate (DSS) Thermo Fisher Scientific CAT#A39267 Critical commercial assays ClonExpress II One Step Cloning Kit Vazyme CAT#C112-02 RNAprep Pure Cell/Bacteria Kit Tiangen CAT#DP430 SuperScript III First Strand kit Invitrogen CAT#18080-051 ChamQ SYBR Color qPCR Master Mix Vazyme CAT#R323-01 Chromatin Immunoprecipitation Kit Millipore CAT#17-371 50 RACE System for Rapid Amplification of cDNA Ends Invitrogen CAT#18374058 Pyruvate concentration determination kit Sangon Biotech CAT#D799449-0050 Deposited data Genome sequencing files This study GenBank: PRJNA1038743 RNA sequencing files This study GenBank: PRJNA1025119 Experimental models: Cell lines Human lung tissue cell line A549 ATCC Cat#CCL-185 Experimental models: Organisms/strains Mouse: C57BL/6 Shanghai Lingchang Biotechnology CAT#jlc0007 Oligonucleotides See Table S4 for primers used in this study This study N/A Recombinant DNA Plasmid: pACRISPR Chen et al.56 N/A Plasmid: pACRISPR-gavR deletion This study N/A Plasmid: pACRISPR-kan insertion This study N/A Plasmid: pACRISPR-gavR insertion This study N/A Plasmid: pACRISPR-gavR* insertion This study N/A Plasmid: pACRISPR-aceE deletion This study N/A Plasmid: pACRISPR-rpoS deletion This study N/A Plasmid: pHERD20T Chen et al.56 N/A (Continued on next page) Cell Reports 44, 115214, January 28, 2025 17

Techniques: Binding Assay, Sequencing, RNA Sequencing, Standard Deviation, Two Tailed Test, Over Expression, Plasmid Preparation, Control, Infection, Lactate Dehydrogenase Assay