Journal: Virulence

Article Title: LRRC8A promotes Glaesserella parasuis cytolethal distending toxin-induced p53-dependent apoptosis in NPTr cells

doi: 10.1080/21505594.2023.2287339

Figure Lengend Snippet: VRAC blockers inhibit G. parasuis CdtABC-induced apoptosis in NPTr cells. NPTr cells were pre-treated with 30 μM PPQ-102 or 10 μM NS3728 for 2 h and subsequently exposed to 500 ng/mL CdtABC for another 36 h. (a) the percentage of apoptotic cells in NPTr was measured using flow cytometry. (b) after CdtABC treatment, the activity of the apoptosis factor caspase-3 was measured. (c) the expression of p-p53, p53, and BAX protein relative to GAPDH in NPTr cells was analyzed using Western blot. Band intensity ratios were calculated from Western blot, and values are given relative to control cells. The statistical significance of the indicated P values was determined as: * P < 0.05, ** P < 0.01, *** P < 0.001. All data shown are representative of at least three independent experiments.

Article Snippet: The reagents were purchased as follows: Annexin V-fluorescein isothiocyanate (Annexin V-FITC)/propidium iodide (PI) Kit from 4A Biotech (Beijing, China); VRAC inhibitors PPQ-102 and NS3728 from MedChemExpress (Monmouth Junction, NJ, USA), and GreenNuc TM Caspase-3 Assay Kit for Live Cells from Beyotime (Shanghai, China).

Techniques: Flow Cytometry, Activity Assay, Expressing, Western Blot, Control

Journal: Virulence

Article Title: LRRC8A promotes Glaesserella parasuis cytolethal distending toxin-induced p53-dependent apoptosis in NPTr cells

doi: 10.1080/21505594.2023.2287339

Figure Lengend Snippet: The important role of LRRC8A plays in G. parasuis CdtABC-induced apoptosis. the dash line indicates that CdtABC is internalized into the cell followed by the relocation of CdtB to the nucleus through an unknown pathway. As possessing DNase activity, CdtB brings about DNA double-strand breaks (DSB), which leads to DNA damage response. Induction of apoptosis via p53 pathway is generally elicited by CdtABC exposure as DNA damage. Activation of p53 decreased the expression of anti-apoptosis factor Bcl-2 and increased the expression of pro-apoptosis factor BAX, leading to increased mitochondrial outer-membrane permeabilization, cytochrome c release, activation of caspase-9 and subsequent executer caspase-3. LRRC8A is also activated by pro-apoptotic stimuli. Activation of LRRC8A triggers cell shrinkage (apoptotic volume decrease) as Cl – and osmolyte efflux through CdtABC-activated VRAC, which is essential for the phosphorylation of p53 and the progression of apoptosis. Thus, LRRC8A promotes the pro-apoptosis effect of CdtABC.

Article Snippet: The reagents were purchased as follows: Annexin V-fluorescein isothiocyanate (Annexin V-FITC)/propidium iodide (PI) Kit from 4A Biotech (Beijing, China); VRAC inhibitors PPQ-102 and NS3728 from MedChemExpress (Monmouth Junction, NJ, USA), and GreenNuc TM Caspase-3 Assay Kit for Live Cells from Beyotime (Shanghai, China).

Techniques: Activity Assay, Activation Assay, Expressing, Membrane

Journal: bioRxiv

Article Title: Infection dynamics and virulence potential of clinical Pseudomonas aeruginosa isolates in a human airway epithelium model system

doi: 10.1101/2025.04.11.644308

Figure Lengend Snippet: ( ETI) treatment on cystic fibrosis transmembrane conductance protein (CFTR) function and infection pattern in cystic fibrosis (CF) cultures. A) Fold change in TEER measurements after 24 hours of ETI treatement (+ ETI) or no treatement (-ETI) in ALI cultures from three CF donors homozygous for ΔF508 mutation. B) TEEC after CFTR stimulation in treated (+ ETI) and untreated (-ETI) cells. Each dot represents one independent donor. C) TEER change after 14 h of infection with PAO1 laboratory strain in treated (+ ETI) and untreated (-ETI). D) Bacterial growth (CFU) after 14 h of infection in the apical (non attached / free bacteria), attached to the epithelium, and basolateral compartment, along with the total number of bacteria. In A,C-D, each dot represents one independent experiment and the mean ± SD is included. Statistical significance was calculated by paired two-sided t-test where **p<0.01 and ****p<0.0001. E) Three-dimensional reconstruction of confocal microscopy images of immunofluorescent staining of CFTR protein in ALI cultures from the CF donors treated (+ ETI) and untreated (-ETI). Green: Anti-CFTR 570 (CFTR protein), Red: Phalloidin (F-actin), Blue: DAPI (nuclei). ALI: air-liquid interface; TEER: transepithelial electrical resistance; TEEC: transepithelial electrical conductance; CFU: colony-forming units.

Article Snippet: To subsequently inhibit CFTR function, a solution containing CFTR inhibitor PPQ-102 30 μM (TargetMol) was added to the apical side.

Techniques: Infection, Mutagenesis, Bacteria, Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: Expression of Wild-Type CFTR Suppresses NF-κB-Driven Inflammatory Signalling

doi: 10.1371/journal.pone.0011598

Figure Lengend Snippet: A . NF-κB activity in H441 cells transfected with either empty vector or wt-CFTR (400 ng), each treated with 10 µM CFTR inh172 for 4 hours, n = 3 *p<0.05 (compared to empty vector control), # p<0.05 (compared to CFTR control). B . NF-κB activity in 16HBE14o − cells treated with 10 µM CFTR inh172 and/or 10 µM forskolin and 100 µM IBMX for 4 h, n = 7, *p<0.05. C . IL-8 levels in supernatants from 16HBE14o − cells measured by ELISA, n = 8, p not significant. D IL-8 levels in supernatants from primary human nasal epithelial cells treated with 10 µM CFTR inh172 for 4 hours, n = 4, *p<0.05.

Article Snippet: 24 h post transfection, cells were treated with combinations of 10 ng/ml TNFα, to stimulate NF-κB activity, 10 µM CFTR inh172 (Merck, Nottingham, U.K.), to inhibit CFTR and 10 µM Forskolin and 10 µM IBMX to stimulate PKA by elevating cAMP.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Effects of Akt Activator SC79 on Human M0 Macrophage Phagocytosis and Cytokine Production

doi: 10.3390/cells13110902

Figure Lengend Snippet: Pharmacological inhibitors and activators used in this study.

Article Snippet: CFTR inh 172 , CFTR inhibitor , Cayman Chemical (15545).

Techniques:

Journal: Cells

Article Title: Effects of Akt Activator SC79 on Human M0 Macrophage Phagocytosis and Cytokine Production

doi: 10.3390/cells13110902

Figure Lengend Snippet: SC79 activates NO production via Akt. ( A ) Bar graph of endpoint DAF-FM fluorescence from 5 independent experiments using macrophages from different donors. Responses were tested with 0.1–10 µg/mL SC79 ± Akt inhibitors MK2206 (10 µg/mL) or GSK690693 (10 µM), PKC inhibitor Gö6983 (10 µM), PKA inhibitor H89 (10 µM), NOS inhibitor L-NAME (10 µM), or inactive D-NAME (10 µM). Significance was determined via one-way ANOVA with Dunnett’s post-test, comparing values to those for HBSS alone; * p < 0.05. ( B ) DAF-FM fluorescence data with 1 and 10 µg/mL SC79 ± 10 µM CFTR inh 172 pretreatment. No significant differences were determined via one-way ANOVA. ( C ) Representative real-time traces of DAF-FM fluorescence, ± L-NAME or D-NAME. Time of addition of the indicated drugs is denoted by the arrow. ( D ) Data from 5 independent experiments done similarly as in ( C ). Significance was determined via one-way ANOVA with Dunnett’s post-test, comparing values to those for SC79 alone; * p < 0.05.

Article Snippet: CFTR inh 172 , CFTR inhibitor , Cayman Chemical (15545).

Techniques: Fluorescence

Journal: Cells

Article Title: Effects of Akt Activator SC79 on Human M0 Macrophage Phagocytosis and Cytokine Production

doi: 10.3390/cells13110902

Figure Lengend Snippet: SC79 enhancement of phagocytosis is not altered by CFTR inh 172. ( A ) The same type of FITC E. coli phagocytosis experiments as in , testing the SC79 ± CFTR inh 172 pretreatment. ( B ) The same type of phagocytosis experiments of pHrodo S. aureus as in , but testing SC79 ± CFTR inh 172 pretreatment. Significance was determined via one-way ANOVA with Bonferroni’s post-test with paired comparisons; * p < 0.05 vs. 0 µg/mL SC79 (HBSS + 0.1% DMSO vehicle control); n.s. means there was no statistical significance between bracketed groups. Data from 5–6 independent experiments per condition with macrophages from different donors.

Article Snippet: CFTR inh 172 , CFTR inhibitor , Cayman Chemical (15545).

Techniques: Control