4 pba  (TargetMol)

Journal: Veterinary Research

Article Title: Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade

doi: 10.1186/s13567-025-01679-6

Figure Lengend Snippet: APEC OMVs induce ERS in HD11 cells to evade immune clearance by macrophages . Transmission electron microscopy shows ER network expansion and swelling in HD11 cells treated with WT, WTΔ ypjA , or OMVs. The data represent one of three independent experiments. Scale bar, 2 μm. B OMVs colocalized with ER. DiO-OMVs were incubated with HD11 cells at 37 °C for 6 h, the ER was labeled with ER-Tracker Red, and laser confocal microscopy imaging was performed. The data represent one of three independent experiments. Scale bar, 7.5 μm. Colocalization scatter plot was generated using ImageJ software and GraphPad Prism 8. C HD11 cells were treated with 50–200 µg/mL OMVs for 6 h, and the relative mRNA level of GRP78/BiP was determined by qPCR. ( n = 3) D HD11 cells were treated with OMVs (100 µg/mL) for 0–9 h, and the relative mRNA level of GRP78/BiP was determined by qPCR. ( n = 3) E , F HD11 cells were treated with OMVs (100 µg/mL) for 0–9 h, and western blot was used to detect the expression of GRP78/BiP protein in cell lysates and gray value analysis was performed using ImageJ software ( F ). ( n = 3) G Cell viability of HD11 cells treated with OMVs (200 µg/mL) for 12 h was detected by CCK-8 in the absence or presence of 4-PBA (2 mM). ( n = 3) H , I Intracellular survival of HD11 cells infected with WT at MOI = 100 in the absence or presence of 4-PBA (2 mM). Square culture plates showed 1/ 100 of the bacterial load. ( n = 3). n represents three biological replicates; data points indicate independent culture systems. Bar charts display mean ± standard error of the mean (SEM). Data were analyzed using Student’s t -test and two-way ANOVA with Sidak correction. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: 4-Phenylbutyric acid (4-PBA) , MedChemExpress, Shanghai, China , 2 mM.

Techniques: Transmission Assay, Electron Microscopy, Incubation, Labeling, Confocal Microscopy, Imaging, Generated, Software, Western Blot, Expressing, CCK-8 Assay, Infection

Journal: Veterinary Research

Article Title: Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade

doi: 10.1186/s13567-025-01679-6

Figure Lengend Snippet: APEC OMVs activate UPR signaling to induce ERS . A HD11 cells were treated with 50–200 µg/mL OMVs for 6 h, and the relative mRNA levels of UPR downstream genes ATF4 , CHOP , EDEM1 , ERdj4 , and ATF6 were detected by qPCR. ( n = 3) B HD11 cells were treated with OMVs (100 µg/mL) for 0–9 h, and qPCR was used to detect the relative mRNA levels of UPR downstream genes ATF4 , CHOP , EDEM1 , ERdj4 , and ATF6 . ( n = 3) C , D HD11 cells were treated with OMVs (100 µg/mL) for 0–9 h, western blotting was used to detect the expression of PERK, p-PERK, eIF2α, p-eIF2α, CHOP, IRE1, p-IRE1, and ATF6 proteins in cell lysates, and gray value analysis was performed using ImageJ software ( D ). ( n = 3). E , F HD11 cells were treated with OMVs (100 µg/mL) for 6 h in the absence or presence of 4-PBA (2 mM), western blot analysis was used to detect the expression of PERK, p-PERK, eIF2α, p-eIF2α, CHOP, IRE1, p-IRE1, and ATF6 proteins in cell lysates, and gray value analysis was performed using ImageJ software ( F ). ( n = 3); n represents three biological replicates; data points indicate independent culture systems. Bar charts display mean ± standard error of the mean (SEM). Two-way ANOVA with Sidak correction was performed (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: 4-Phenylbutyric acid (4-PBA) , MedChemExpress, Shanghai, China , 2 mM.

Techniques: Western Blot, Expressing, Software

Journal: Veterinary Research

Article Title: Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade

doi: 10.1186/s13567-025-01679-6

Figure Lengend Snippet: APEC OMVs activate ERS to inhibit autophagosome degradation. A , B HD11 cells were treated with OMVs (100 µg/mL) for 6 h in the absence or presence of 4-PBA (2 mM), western blot analysis was used to detect the expression of LC3-II protein in cell lysates, and gray value analysis was performed using ImageJ software ( B ) ( n = 3). C , D HD11 cells were treated with OMVs (100 µg/mL) for 6 h in the absence or presence of 4-PBA (2 mM). LC3 dot aggregation was detected by immunofluorescence and statistically analyzed ( D ). Data represent one of three independent experiments. Scale bar, 5 µm. E HD11 cells transiently expressing the mCherry-GFP-LC3 plasmid were co-incubated with OMVs (100 µg/mL) for 6 h in the absence or presence of 4-PBA (2 mM) or Mito-TEMPO (10 µM), with CQ (10 µM) and bafA1 (100 nM) as controls. Fluorescence intensity of mCherry and GFP was detected in cells using laser confocal microscopy. Data represent one of three independent experiments; scale bar, 5 μm. Colocalization scatter plots were generated using ImageJ software and GraphPad Prism 8. F HD11 cells were treated with OMVs (100 µg/mL) for 6 h in the absence or presence of 4-PBA (2 mM), with CQ (10 µM) as a control. Fluorescence intensity of LysoSensor and LysoTracker in cells was detected by laser confocal microscopy. Data represent one of three independent experiments. Scale bar, 5 μm. Colocalization scatter plots were generated using ImageJ software and GraphPad Prism 8. represents three biological replicates; data points represent independent cultures. Bar charts show mean ± standard error of the mean (SEM); analysis performed using Student’s t -test (*** p < 0.001).

Article Snippet: 4-Phenylbutyric acid (4-PBA) , MedChemExpress, Shanghai, China , 2 mM.

Techniques: Western Blot, Expressing, Software, Immunofluorescence, Plasmid Preparation, Incubation, Fluorescence, Confocal Microscopy, Generated, Control

Journal: Veterinary Research

Article Title: Outer membrane vesicles secreted by avian pathogenic Escherichia coli promote its survival within macrophages and systemic infection by inducing endoplasmic reticulum stress-mediated autophagy flux blockade

doi: 10.1186/s13567-025-01679-6

Figure Lengend Snippet: APEC OMVs induce ERS to promote APEC systemic infection . Flowchart of the infection experiment using chicks treated with 4-PBA. B On the sixth day after WT infection of chicks (1 × 10 CFU/chick), the number of bacteria (CFU) was counted in the trachea, lungs, liver, and spleen tissues of the chicks in the absence or presence of 4-PBA (50 mg/kg). Trachea: 95% CI [0.26–0.38]; lungs: 95% CI [0.65–0.78]; liver: 95% CI [0.004–0.13]; spleen: 95% CI [0.08–0.21]. C On the sixth day after WT infection of chicks (1 × 10 9 CFU/chick), HE staining was performed on tracheal, lung, liver, and spleen tissues of the chicks in the absence or presence of 4-PBA (50 mg/kg). Trachea: degeneration and hyperplasia of epithelial mucosal cells (black arrow), diffuse neutrophilic infiltration (red arrow), mucus and inflammatory exudate in the mucosa of the tracheal wall (blue arrow); lung: markedly widened pulmonary septa (black arrow), inflammatory cell infiltration in the pulmonary interstitium (red arrow); liver: hepatocytes show lytic degeneration (black arrow) with inflammatory cell infiltration (red arrow); spleen: marked hyperplasia of lymphoid follicles (black arrow), blurred demarcation between white and red pulp (red arrow). Scale bar, 50 µm or 200 µm. D – F GRP78/BiP immunofluorescence staining was performed on lung and spleen tissues from chicks on day 6 post-infection ( D ) and analyzed using ImageJ software ( E , F ). Scale bar, 50 µm. Data points represent independent cultures; bar charts show mean ± standard error of the mean (SEM). Two-way ANOVA with Sidak correction was performed (* p < 0.05, ** p < 0.01, *** p < 0.001). Effect sizes with 95% confidence intervals are reported in panel ( B ).

Article Snippet: 4-Phenylbutyric acid (4-PBA) , MedChemExpress, Shanghai, China , 2 mM.

Techniques: Infection, Bacteria, Staining, Immunofluorescence, Software