Journal: ACS chemical biology

Article Title: Fluorescence Assessment of the AmpR-Signaling Network of Pseudomonas aeruginosa to Exposure to β ‑Lactam Antibiotics

doi: 10.1021/acschembio.9b00875

Figure Lengend Snippet: Schematic depiction of reporter plasmid and fluorescence gene induction by the β-lactam antibiotic. (A) The plasmid map for pCN61_AmpRtdT is shown to the left. Ampicillin (AP, Tem-1 β-lactamase) or streptomycin (STR) is used for selection. The ampRC operon (top right) and the reporter operon where tdTomato is positioned downstream of the ampC promoter (denoted PampC) are depicted. (B) We constructed GFP-labeled P. aeruginosa and transformed this bacterium with pCN61_AmpRtdT. The bacteria were inoculated on an agar plate in proximity to the β-lactam antibiotic CAZ (spotted at the red dot). The antibiotic diffuses outward from the spot. The interface (white arrow) of CAZ and the GFP-labeled P. aeruginosa without the plasmid shows only green fluorescence (top row). In contrast, the GFP-labeled P. aeruginosa transformed by pCN61_AmpRtdT shows green fluorescence (constituitively) and the red fluorescence induced by the cell-wall-acting antibiotic (bottom row). The top row confirms the absence of red backgroud fluorescence from P. aeruginosa. The bottom row demonstrates the functionality of our reporter plasmid. A 10 μM scale bar is given in panel two of the top row.

Article Snippet: The fluorescent-reporter systems were excised from pUC57 with restriction enzymes Bam HI and Pst I and ligated into an E. coli - P. aeruginosa shuttle vector, pCN61 (ATCC).

Techniques: Plasmid Preparation, Fluorescence, Selection, Construct, Labeling, Transformation Assay

Journal: ACS chemical biology

Article Title: Fluorescence Assessment of the AmpR-Signaling Network of Pseudomonas aeruginosa to Exposure to β ‑Lactam Antibiotics

doi: 10.1021/acschembio.9b00875

Figure Lengend Snippet: Correlation of the β-lactamase and fluorescence assays. (A) Nitrocefin hydrolysis by AmpC β-lactamase shifts λmax from 390 nm (yellow) to 486 nm (red). (B) Nitrocefin assay performed for P. aeruginosa wild-type and dacB::Tn is plotted as the slope of the absorbance at 486 nm over time for two concentrations of FOX (1/8 MIC and 1/4 MIC) and no antibiotic. (C) Fluorescent response expressed as relative fluorescence (A.U.) of P. aeruginosa wild-type and dacB::Tn under the same experimental conditions as used for the nitrocefin assay. The error bars represent the standard deviation of three biological replicates.

Article Snippet: The fluorescent-reporter systems were excised from pUC57 with restriction enzymes Bam HI and Pst I and ligated into an E. coli - P. aeruginosa shuttle vector, pCN61 (ATCC).

Techniques: Fluorescence, Standard Deviation

Journal: ACS chemical biology

Article Title: Fluorescence Assessment of the AmpR-Signaling Network of Pseudomonas aeruginosa to Exposure to β ‑Lactam Antibiotics

doi: 10.1021/acschembio.9b00875

Figure Lengend Snippet: GFP-labeled P. aeruginosa harboring pCN61_AmpRtdT was imaged on a swarm plate in proximity of a second bacterium, either (A) P. mesacidophila, (B) B. licheniformis, (C) E. coli, or (D) M. xanthus. Each panel depicts the bacteria after 15 h of growth. P. aeruginosa is to the left of each plate, while the second bacterium is to the right (see plate panels). The white arrow (top plate) points to a representative site where the two bacteria encounter one another. The second column from left shows bright-field images of comingled bacteria, where the strains meet, as uniform continuous lawns. The GFP panel shows the green fluorescence displayed by P. aeruginosa in this lawn. The black voids are the locations of the second bacterium in this same lawn. The RFP panel shows the red-fluorescent signal from P. aeruginosa that results from contact with an antibiotic-producer strain. Red fluorescence is seen for P. mesacidophila and B. lichemiformis as antibiotic producers. No red fluorescence is seen for E. coli and M. xanthus (not antibiotic producers). The far-right column merges the green and red fluorescent images. A 10-μm black scale bar is given in the bright-field image of panel A.

Article Snippet: The fluorescent-reporter systems were excised from pUC57 with restriction enzymes Bam HI and Pst I and ligated into an E. coli - P. aeruginosa shuttle vector, pCN61 (ATCC).

Techniques: Labeling, Fluorescence

Journal: Frontiers in Oncology

Article Title: E2F1 mediates competition, proliferation and response to cisplatin in cohabitating resistant and sensitive ovarian cancer cells

doi: 10.3389/fonc.2024.1304691

Figure Lengend Snippet: Long term co-culture of platinum-sensitive and resistant cells increases proliferation and sensitivity to platinum of sensitive cells. (A, B) Pictures of co-cultured PE01 and PE04 (A) and OVCAR5 WT and OVCAR5 CisR (B) ovarian cancer cells transduced with RFP or GFP lentiviral vectors. Images show cells at day 5 of co-culture at the ratios of 1:5 (sensitive to resistant) (2 replicates each, scale bar: 200 µm). (C, D) Cell proliferation (mean ± SD, n=3) measured with CCK8 assays in PE01 and PE04 (C) and OVCAR5 WT and CisR (D) cells maintained in co-culture (CC) or monoculture (MC). Cells were co-cultured for 14 days, sorted by FACS and then cultured to evaluate cell viability. Blue asterisk indicates statistical significance for the comparison between sensitive cells, marron asterisk indicates statistical significance for the comparison between resistant cells (E-H) Representative curves of cisplatin effects on cell viability of PE01 (E) , PE04 (F) , OVCAR5 WT (G) and OVCAR5 CisR (H) maintained in co-culture as described in (A, B) vs. the same cell lines in monoculture. (I, J) Comparison of cisplatin IC 50 values (means ± SD, n=3) between monocultured and co-cultured PE01 with PE04 (I) and OVCAR5 WT with CisR cells (J) . All graphs are representative of 3 independent replicates. *p<0.05; **p<0.01.

Article Snippet: GFP and RFP stable PE01, PE04 and OVCAR5 cells were generated by cell transduction with lentiviral vectors (Gentarget #LVP001 and #LVP023, respectively) followed by selection with puromycin and flow cytometry-based cell sorting.

Techniques: Co-Culture Assay, Cell Culture, Transduction, Comparison