Journal: Veterinary Research
Article Title: RfeA from Streptococcus suis serotype 2 triggers NLRP3/Caspase-1-dependent pyroptosis leading to blood–brain barrier disruption
doi: 10.1186/s13567-025-01620-x
Figure Lengend Snippet: RfeA activates the NLRP3/Caspase-1 pathway through mtROS . A Representative western blots of the NLRP3 and Caspase-1 p20 proteins in hBMECs pretreated with CA-074me, AZ10606120, or MitoTempo followed by 4 h of rRfeA stimulation. β-Tubulin served as a loading control. B Quantitative analysis of NLRP3 and Caspase-1 p20 intensities in panel A. Vehicle-treated cells were used as a reference (1.0). The data are presented as the means ± s.d.; n = 3. Significant differences compared with no inhibitor were identified by two-way ANOVA followed by Dunnett’s multiple comparisons test. ** P < 0.01; *** P < 0.001. C ELISA quantification of IL-1β secretion in culture supernatants from hBMECs pretreated with inhibitors (CA-074me, AZ10606120, MitoTempo) prior to 4 h of rRfeA stimulation. The data are presented as the means ± s.d.; n = 3. Significant differences compared with no inhibitor were identified by one-way ANOVA followed by Dunnett’s multiple comparisons test. *** P < 0.001. D Confocal microscopy images depicting mtROS accumulation in hBMECs at 0, 2, and 4 h post-rRfeA stimulation subjected to mitoSOX staining. Scale bar: 5 μm. E Quantitative analysis of the mean fluorescence intensity of mitoSOX. X-axis: different incubation times; Y-axis: MFI (arbitrary units). The data are presented as the means ± s.d.; n = 52 images. Significant differences compared with 0 h were identified by one-way ANOVA followed by Dunnett’s multiple comparisons test. *** P < 0.001. F Flow cytometric analysis of mtROS levels in hBMECs at 0, 2, and 4 h postrRfeA stimulation after mitoSOX staining.
Article Snippet: For experiments involving chemical inhibitors, hBMECs were pretreated separately with chlorpromazine (Selleck, 15 μg/mL, 1 h), wortmannin (MCE, 200 nM, 1 h), calmidazolium (MCE, 10 μM, 1 h), glibenclamide (MCE, 100 μM, 0.5 h), Z-YVAD-FMK (MCE, 5 μM, 0.5 h), CA-074me (MCE, 10 μM, 1 h), AZ10606120 (MCE, 10 μM, 1 h), Mito Tempo (MCE, 250 μM, 1 h), or VBIT-12 (MCE, 10 μM, 6 h).
Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Staining, Fluorescence, Incubation
