Journal: PNAS Nexus

Article Title: Bacterial membrane vesicles of Pseudomonas aeruginosa activate adenosine monophosphate-activated protein kinase signaling through inhibition of mitochondrial complex III

doi: 10.1093/pnasnexus/pgaf248

Figure Lengend Snippet: Metabolic reprogramming in host cells after PA14 BMV treatment. A) Confluence of A549 and HCC44 cells after treatment with 25 VL/mL BMVs for 72 h. Data were obtained from three replicates. B) Glucose uptake and lactate secretion of A549, HCC44, and HBEpC cells after treatment with 25 VL/mL BMVs for 24 h. Data were obtained from three replicates. C) Signal intensities (AU) of malate, aspartate, and glutamate in A549 and HBEpC cells after vesicle treatment (25 VL/mL for 24 h). Data were obtained from 5 to 6 (A549) or 2 to 3 (HBEpC) replicates and normalized to cell confluence. D) Schematic overview of the incorporation of a [U- 13 C 6 ]-glucose tracer into metabolites of the TCA cycle. Created with BioRender.com . E) Malate MIDs after [U- 13 C 6 ]-glucose labeling of BMV-treated cells. A549 and HCC44 cells were treated for 24 h and HBEpC cells for 6 h with 25 VL/mL. Data were obtained from two or three replicates. F) Schematic overview of the incorporation of a [U- 13 C 5 ]-glutamine tracer into metabolites of the TCA cycle. Created with BioRender.com . G) Malate MIDs after [U- 13 C 5 ]-glutamine labeling of BMV-treated cells. A549 and HCC44 cells were treated for 24 h and HBEpC cells for 6 h with 25 VL/mL. Data were obtained from three replicates. All bar plots in this figure are depicted as mean ± SEM. For the comparison between control (Ctrl) and BMV-treated cells for each cell line, statistical significance was analyzed using unpaired t test (n.s. = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: HBEpC cells were cultivated in Airway Epithelial Cell Growth Medium supplemented with Growth Medium SupplementMix (PromoCell, C-21060).

Techniques: Labeling, Comparison, Control

Journal: PNAS Nexus

Article Title: Bacterial membrane vesicles of Pseudomonas aeruginosa activate adenosine monophosphate-activated protein kinase signaling through inhibition of mitochondrial complex III

doi: 10.1093/pnasnexus/pgaf248

Figure Lengend Snippet: PA14 BMVs affect transcription and translation of the host cell. A) RNA-sequencing analysis of A549 cells treated with PA14 BMVs (PA) for 24 h. B) Relative gene expression of HMGCR and SREBF2 in A549 cells after treatment with 25 VL/mL BMVs for 24 h. Data were obtained from three replicates. C) IL-8 secretion of A549 and HBEpC cells after BMV treatment (25 VL/mL for 24 h). Data were obtained from three replicates. D) Global protein synthesis of A549 cells after treatment with 50 µg/mL CHX and 25 VL/mL BMVs for 30 min (+30 min pretreatment). Data were obtained from four or five replicates. All bar plots in this figure are depicted as mean ± SEM. Statistical significance was analyzed using unpaired t test (B, C) or using unpaired one-way ANOVA followed by Tukey's multiple comparison test (D) (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: HBEpC cells were cultivated in Airway Epithelial Cell Growth Medium supplemented with Growth Medium SupplementMix (PromoCell, C-21060).

Techniques: RNA Sequencing, Gene Expression, Comparison

Journal: PNAS Nexus

Article Title: Bacterial membrane vesicles of Pseudomonas aeruginosa activate adenosine monophosphate-activated protein kinase signaling through inhibition of mitochondrial complex III

doi: 10.1093/pnasnexus/pgaf248

Figure Lengend Snippet: Activation of AMPK signaling in host cells by PA14 BMVs. A) Overview of the AMPK signaling targets ACC1 and eEF2 and their activation. B) Western blot (WB) analysis of p-ACC1 (Ser79) in A549 cells after vesicle treatment (25 and 50 VL/mL) for 24 h. Data were obtained from two independent experiments. C) WB analysis of p-eEF2 (Thr56) in A549 cells after vesicle treatment (25 and 50 VL/mL) for 24 h. Data were obtained from two independent experiments. D) WB analysis of p-eEF2 (Thr56) in HCC44 cells after vesicle treatment (25 and 50 VL/mL) for 24 h. E) WB analysis of p-eEF2 (Thr56) in HBEpC cells after vesicle treatment (25 and 50 VL/mL) for 24 h. All bar plots in this figure are depicted as mean (±SEM).

Article Snippet: HBEpC cells were cultivated in Airway Epithelial Cell Growth Medium supplemented with Growth Medium SupplementMix (PromoCell, C-21060).

Techniques: Activation Assay, Western Blot