Selective infection of ACPP-pc-Ad-eGFP in MMP-overexpressing cells and control.

<p>HBE(control), A549, MDA-MB-231 and HepG2 cells were seeded into 96-well plates (10⁴ cells per well) and after 24 h were infected with 10⁴ particles per cell of ACPP-pc-Ad-eGFP in DMEM/10% FCS (A549, MDA-MB-231) or RRPMI-1640/10% FCS (HBE, HepG2). The supernatant was removed 4 h after infection and incubated with 200 µl DMEM/10% FCS (A549, MDA-MB-231) or RRPMI-1640/10% FCS (HBE, HepG2) for an additional 48 h before fluorescence was measured. The columns depict the following: i, HBE; ii, A549; iii, MDA-MB-231; and iv, HepG2. Data are the means ± SEM. *P<0.05 compared with the HBE cell.</p

Transduction efficiency of Ad-eGFP, pc-Ad-eGFP and ACPP-pc-Ad-eGFP with A549 cells.

<p>(A) A549 cells were seeded into 96-well plates (10⁴ cells/well) and infected after 24 h incubation with 10⁴ particles per cell of Ad-eGFP, pc-Ad-eGFP and ACPP-pc-Ad-eGFP in DMEM/10% fetal calf serum (FCS). Cellular GFP fluorescence was visualized 48 h post-infection using a Nikon TI-S microscope and photographed with a Nikon camera. (i) Uninfected cells; infection with (ii) Ad-eGFP, (iii) pc-Ad-eGFP, and (iv) ACPP-pc-Ad-eGFP. (B) After A549 cells were infected for 48 h as described above, the medium was removed, the cells lysed with 100 ml Triton X-100 (0.2% in H₂O) and GFP fluorescence was measured (λ_ex 488 nm and λ_em 538 nm) with a Fluoroskan fluorescence plate reader (Multiskan GO, Thermo Scientific). The columns depict the following: (i) uninfected cells, (ii) Ad-eGFP, (iii) pc-Ad-eGFP, and (iv) ACPP-pc-Ad-eGFP. Data are the means ± SEM. *P<0.05 compared with i, ^#P<0.05 compared with iii (C) A549 cells were trypsinized, aliquoted at (2×10⁵ cells)/(2 ml DMEM/10% FCS) and incubated in 6-well plates at 37°C until 90% confluence was reached; subsequently, 10⁹ particles Ad-eGFP, pc-Ad-eGFP or ACPP-pc-Ad-eGFP labeled with PI were added. Cells were trypsinized, centrifuged (2 min, 1500 g) and washed in PBS 48 h later. Association of PI-labeled virus with cells was measured using a Coulter EPICS XL flow cytometer with an argon laser (λ_ex 540 nm and λ_em 625 nm). The fluorescence profile of control cells (black line) or cells infected with virus (red line), pc-virus (yellow line) or ACPP-pc-virus (purple line).</p

ACPP-pc-Ad-eGFP distributed in cytoplasm of A549 cells.

<p>(A) cytoplasmic uptake of ACPP marker FITC (green); (B) cytoplasmic uptake of Ad-eGFP marker PI (red); (C) superimposition of the two dyes (green–red staining) confirms ACPP-mediated transport of pc-Ad-eGFP to the cytoplasm. Scale bar represents 100 px.</p

Fluorescence images of A549 cells incubated with ACPP-pc-Ad-eGFP(PI) and pc-Ad-eGFP(PI) after 20 min, 2 h, and 4 h.

<p>Panels A-C depict the cytoplasmic uptake of ACPP-pc-Ad-eGFP at 20 min (A), 2 h (B), and 4 h (C). Panels d-f depict the cytoplasmic uptake of pc-Ad-eGFP at 20 min (D), 2 h (E), and 4 h (F). Scale bar represents 100 px.</p

FITC fluorescence of ACPP in A549, MDA-MB-231, HepG2 and HBE.

<p>(A-C) A549, MDA-MB-231, HepG2 showed high FITC fluorescence of ACPP SP×200. (D) HBE showed low FITC fluorescence of ACPP SP×200. (E-H) FITC fluorescence of ACPP in A549, MDA-MB-231, HepG2 and HBE treated with Doxycycline SP×200.</p

Cytoplasmic delivery of ACPP-pc-Ad-eGFP and pc-Ad-eGFP in A549 cells.

<p>Inverted fluorescence microscopy image show(A, B) cytoplasmic marker CFSE (green), (C) incubated ACPP-pc-Ad-eGFP(PI) with A549 cells for 4 h at 37°C yielded uptake of the conjugate (red); (D) incubation pc-Ad-eGFP(PI) with A549 cells for 4 h at 37°C yielded little uptake (red); (E) superimposition of the two dyes (A and C) confirms ACPP-mediated nonendocytotic transport to the cytoplasm. (F) superimposition of the two dyes (Band D) exhibited only endocytotic vesicles. Scale bar represents 100 px.</p

Virus neutralization assay of Ad-eGFP or ACPP-pc-Ad-eGFP.

<p>Ad-eGFP or ACPP-pc-Ad-eGFP (10⁸ particles per 100 µl) was incubated with A549 cells in a 96-well plate (10⁴ cells per well; 10⁴ virus particles per cell) in the presence or absence of neutralizing antibodies (NAb). Expression of GFP was measured at 48 h after Triton X-100 lysis and was expressed as a percentage of the fluorescence signal in the absence of human serum. The yellow bar indicates Ad-eGFP, while the black bar denotes ACPP-pc-Ad-eGFP. Data are the means ± SEM. *P<0.05 compared with Ad-eGFP.</p

Protein of MMP-2 and MMP-9 in cell lines HepG2, HBE, A549 and MDA-MB-231 (n = 3).

<p><i>Left</i>, a representative Western blot; <i>Right</i>, densitometric analysis of the representative Western blot, <i>bars</i> represent the relative amounts of MMP-2 and MMP-9. Data are the means ± SEM. *P<0.05 compared with the HBE cell.</p

MOESM1 of LesR is a novel upstream regulator that controls downstream Clp expression to modulate antibiotic HSAF biosynthesis and cell aggregation in Lysobacter enzymogenes OH11

Additional file 1. Table S1. Primers used in this study. Table S2. Genes controlled by LesR in Lysobacter enzymogenes. Figure S1. LeDSF signaling did not control the Clp protein level

Comment refonder une cité coloniale ? Représentation discursive du temps et régime d'historicité.

Additional file 1: Table S1. Confirmation of single mutation by PCR in this study. Table S2. Validation of double or triple mutation by PCR in this study. Figure S1. Lsp proteins contributed to the growth pattern of Lysobacter enzymogenes. Figure S2. Genomic organization of three Lsp-coding genes in Lysobacter enzymogenes