thp1 null2 cells  (InvivoGen)

Journal: mSystems

Article Title: Exploiting gasdermin-mediated pyroptosis for enhanced antimicrobial activity of phage endolysin against Pseudomonas aeruginosa

doi: 10.1128/msystems.01106-24

Figure Lengend Snippet: The experimental pipeline of the study . Recombinant gasdermin D (GSDMD) or its N-terminal region for membrane permeability of P. aeruginosa PAO1 and its LPS O-chain-deficient mutant ( ΔwbpL ) was monitored using NPN uptake assay. Membrane permeability experiments were performed for GSDMDs alone and in the presence of KP27 endolysin . Mimicking bacterial infection—gasdermin D activation by P. aeruginosa O10 LPS in A549, HeLa, or THP1 cells was measured by microarray (gene expression) and ELISA (IL-1β) assays . The inflammatory properties of LPS isolated from E.coli (control) and tested P. aeruginosa strains were evaluated using immune-serological tests. The patterns of isolated LPS samples (the O-chain presence or absence) were characterized using the SDS-PAGE method . LPS-induced pyroptosis alone or in combination with nigericin was tested on the THP1 cell line to obtain active gasdermin D for enhanced KP27 endolysin activity against P. aeruginosa PAO1 strain. LPS was isolated from tested P. aeruginosa strains and E. coli (control). Gasdermin (GSDMD) production was monitored using Western blot, and the antibacterial activity of endolysin was evaluated using culture optical density (OD 600 ).

Article Snippet: A549 cells (ATCC, Manassas, Virginia, CCL-185), BEAS-2B cells (ATCC, Manassas, Virginia, CRL-9609), HeLa cells (ATCC, Manassas, Virginia, CCL-2), THP1-Xblue cells (Invivogen, Toulouse, France), and THP1-Null2 cells (Invivogen, Toulouse, France) were used in our study.

Techniques: Recombinant, Membrane, Permeability, Mutagenesis, Infection, Activation Assay, Microarray, Expressing, Enzyme-linked Immunosorbent Assay, Isolation, Control, SDS Page, Activity Assay, Western Blot

Journal: mSystems

Article Title: Exploiting gasdermin-mediated pyroptosis for enhanced antimicrobial activity of phage endolysin against Pseudomonas aeruginosa

doi: 10.1128/msystems.01106-24

Figure Lengend Snippet: The induction of pyroptosis in monocytic cells by P. aeruginosa O10 LPS. The GSDMD expression level analysis by RT-qPCR in THP1-Xblue cells after treatment with serial concentrations of O10 LPS. The ACTB gene was used as an internal reference gene, and the ΔΔCT method was applied for relative quantification (* P < 0.05) ( A ). The IL-1β production by THP1-Xblue cells in the presence of P. aeruginosa O10 LPS measured using ELISA; * P < 0.05 ( B ). One-way ANOVA with Dunnett’s post-hoc test was used to test the statistically significant differences.

Article Snippet: A549 cells (ATCC, Manassas, Virginia, CCL-185), BEAS-2B cells (ATCC, Manassas, Virginia, CRL-9609), HeLa cells (ATCC, Manassas, Virginia, CCL-2), THP1-Xblue cells (Invivogen, Toulouse, France), and THP1-Null2 cells (Invivogen, Toulouse, France) were used in our study.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: mSystems

Article Title: Exploiting gasdermin-mediated pyroptosis for enhanced antimicrobial activity of phage endolysin against Pseudomonas aeruginosa

doi: 10.1128/msystems.01106-24

Figure Lengend Snippet: The OD 600 of P. aeruginosa PAO1 culture growth was measured in TSB supplemented with 2× diluted THP1-Null2 cell line supernatant, either alone or with 36 µg/mL of KP27 endolysin ( E ) after 18 h of incubation at 37°C. The THP1-Null2 line was first pre-treated with 1 µg/mL of LPS ( S ) and/or 10 µM nigericin ( N ). C, bacterial culture treated with THP1-Null2 supernatant; C E , bacterial culture treated with THP1-Null2 supernatant and endolysin; C N , bacterial culture treated with THP1-Null2 supernatant after nigericin stimulation; C EN , bacterial culture treated with THP1-Null2 supernatant after nigericin stimulation and supplemented with endolysin; S, bacterial culture treated with THP1-Null2 supernatant after LPS stimulation; S E , bacterial culture treated with THP1-Null2 supernatant after LPS stimulation and supplemented with endolysin; S N , bacterial culture treated with THP1-Null2 supernatant after LPS and nigericin stimulation; S EN , bacterial culture treated with THP1-Null2 supernatant after LPS and nigericin stimulation and supplemented with endolysin. Color columns show particular LPS samples used for pyroptosis induction. The experiments were done in three biological repeats, and statistical data were established by one-way ANOVA with post-hoc Dunnett’s test. The P -values are presented in the bottom table.

Article Snippet: A549 cells (ATCC, Manassas, Virginia, CCL-185), BEAS-2B cells (ATCC, Manassas, Virginia, CRL-9609), HeLa cells (ATCC, Manassas, Virginia, CCL-2), THP1-Xblue cells (Invivogen, Toulouse, France), and THP1-Null2 cells (Invivogen, Toulouse, France) were used in our study.

Techniques: Incubation