Z yvad fmk

Human fetal primary RPE cells (pRPE) were obtained from Lonza (Cologne, Germany), cultured as recommended by the manufacturer, and used in experiments for a maximum of six passages. The human nontransformed RPE cell line ARPE-19 (CRL-2302; ATCC, Rockville, MD, USA) was cultured as previously reported [16 (link)]. The immortalized murine wild-type and NLRP3 knockout macrophage cell lines used in our study have been characterized previously [13 (link)] and were cultured as described [17 (link)].
For inhibition of photooxidative damage, the singlet oxygen scavenger 1,4-diazabicyclooctane (DABCO; Sigma-Aldrich, Munich, Germany) was added to the culture media at a concentration of 30 mM during blue light irradiation. Lysosomal membrane permeabilization was induced by incubation of cells with 1 mM ciprofloxacin (Sigma-Aldrich, Munich, Germany) for 3 h or 1 mM l-leucyl-l-leucine methyl ester (Leu-Leu-OMe; Bachem, Bubendorf, Switzerland) for 3 h. Cathepsin B inhibitor CA-074 (Calbiochem, Darmstadt, Germany) and cathepsin L inhibitor Z-Phe-Phe-fluoromethylketone (Z-FF-FMK; Calbiochem) were used for cell treatment at a concentration of 10 μM each for 1 h prior to and during irradiation treatment. For inhibition of caspase-1, we applied 10 μM of Z-Tyr-Val-Ala-Asp-fluoromethylketone (Z-YVAD-FMK; BioVision, Munich, Germany) 30 min prior to and during irradiation.

BV2 cells, a murine microglial cell line, were obtained from the Cell Culture Center Xiehe Medical University (Beijing, China) and cultured in a humidified incubator at 37°C with 5% CO₂ in RPMI 1640 (Gibco, Grand Island, NY, United States) supplemented with 10% heat-inactivated FBS (Gibco), 100 μg/ml streptomycin, and 100 U/ml penicillin (Gibco).
To induce synthesis of pro-IL-1β, mimicking the chronic activation of microglia in prion disease, BV2 cells were primed with 300 ng/ml LPS for 3 h before treatment with PrP106-126. Priming with LPS was necessary because pro-IL-1β is not constitutively expressed in microglia (Dostert et al., 2008 (link)).
For 3-MA (2 mM) (Sigma) and rapamycin (100 nM) (Beyotime Biotechnology) treatment, BV2 cells were primed with 300 ng/ml LPS for 3 h, and were then incubated with PrP106-126 alone, PrP106-126 + 3-MA, or PrP106-126 + rapamycin.
For the treatment inhibiting of caspase-1, BV2 cells were primed with 1 μl/ml of inhibitor Z-YVAD-FMK (Biovision, San Francisco, CA, United States) for 2 h, and were then treated with PrP106-126.

Poly(I:C) was obtained from InvivoGen (San Diego, CA). caspase-1 inhibitor (Z-YVAD-FMK) and caspase-4 inhibitor (Z-LEVD-FMK) were purchased from BioVision (Milpitas, CA). For immunohistochemical analysis and western blotting, we used the following specific antibodies: IL-1β and HMGB1 (Abcam, Cambridge, MA), caspase-1 (Cell Signaling Technology, Danvers, MA), NLRP3 and ASC (Adipogen, San Diego, CA), and actin, α-tubulin and laminB (Santa Cruz Biotechnologies, Santa Cruz, CA).

Blocking antibodies anti-Dectin-1 (2A11), anti-Dectin-2 (D2.11E4) and anti-CR3 (5C6) were purchased from Serotec and anti-TLR-2 (6C2), IgG2a (eBR2a) and IgG2b (eB149/10H5) were from eBioscience. PE/Cy7-conjugated anti-CD45 (30-F11), APC-conjugated anti-F4/80 (BM8), Alexa 488-conjugated anti-CD103 (2E7) and Alexa 647-conjugated anti-Ly6G (1A8) were obtained from BioLegend. APC-conjugated anti-CD11c (N418) and PE-conjugated anti-CD11b (M1/70) were from eBioscience and PE-conjugated anti-Siglec-F (E50-2440) from Pharmingen.
Z-YVAD-FMK (Caspase-1 inhibitor) was obtained from BioVision. U1026 (ERK inhibitor), SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), N-acetyl-L-cysteine (ROS scavenger), Apocynin (NADPH-oxidase inhibitor), Ca-074 methyl ester (cathepsin B inhibitor), bafilomycin A1 (phagosomal acidification inhibitor) and glibenclamide (K⁺ channel inhibitor) were obtained from Sigma-Aldrich.

Porphyromonas gingivalis (P. gingivalis) strain ATCC 33,277 was obtained from the Shanghai Key Laboratory of Stomatology at Shanghai Jiao Tong University School of Medicine. P. gingivalis was grown on anaerobic blood agar plates with 80% N₂, 10% H₂, and 10% CO₂ for 3–5 days. It was then inoculated into brain heart infusion broth supplemented with 5 μg/mL hemin and 1 μg/mL vitamin K for 24 h until reaching an optical density of 0.04 at 660 nm, corresponding to 10⁸ colony-forming units (CFU)/mL. The bacteria were washed and resuspended in α-MEM to infect the PDLSCs at multiplicities of infection (MOIs) of 1:10, 1:50, and 1:100 for 24 h. In some experiments, the PDLSCs were pretreated with Z-VAD-FMK (Sellect, United States), Z-YVAD-FMK (BioVision, United States), Z-LEVD-FMK (BioVision, United States) and DMSO (Sigma, United States) at the indicated concentrations for 1 h before bacterial infection.

C. gariepinus and A. hydrophila (Strain 500,297) were used in the study. The protocols for HKM isolation, and infection of HKM with A. hydrophila (MOI 1:50) have been described earlier [14 (link)].
zHKMs were incubated separately with ER stress inhibitor (4-PBA, 10 µM, Sigma), mtROS inhibitor (YCG063, 10 µM, Calbiochem), ETC inhibitor (antimycin A, 50 µM, Sigma), MPTP inhibitor (Cyclosporin A, 5 µM, Sigma), RyR inhibitor (Dantrolene, 10 µM, Sigma), Cytochalasin D (Cyt D, 5 µg/mL, Sigma), Autophagy inducer (Rapamycin, 20 µM, Sigma), Thapsigargin (1 µM, Sigma), Akt inhibitor (124,005, 10 µM, Calbiochem) for 1 h and mitochondrial Ca²⁺ uptake inhibitor (Ru360, 10 µM, Calbiochem), Caspase-1 inhibitor (Z-YVAD-FMK, 7.5 µM, Biovision), Caspase-3 inhibitor (Z-DEVD-FMK, 10 µM, Biovision), IP3R inhibitor (2-APB, 100 μM, Sigma), PLC inhibitor (U73122, 2 μM, Enzo Life Science), PI3-Kinase inhibitor (LY-294002 hydrochloride, 14.5 μM, Sigma), intracellular Ca²⁺chelator, (BAPTA-AM, 5 mM, Sigma), and JNK inhibitor (SP600125, 10 µM, Calbiochem) for 2 h followed by A. hydrophila infection as mentioned earlier [3 ]. The inhibitor concentrations had no adverse effects on HKM viability and bacterial growth (data not shown).

Antibodies used in the study included rabbit anti-mouse caspase-1 p10 (sc-514) and anti-mouse ASC (sc-22514-R) (Santa Cruz, CA, United States); rabbit anti-mouse NLRP3 (Abcam, Cambridge, MA, United States); rabbit anti-mouse LC3B (MBL, Japan); and rabbit anti-mouse β-actin (Beyotime, Wuhan, Hubei, China); rabbit anti-rat TLR4 (Proteintech, Chicago, IL, United States); rabbit anti-mouse TRIF (Abcam, Cambridge, MA, United States); rabbit anti-mouse GAPDH (Proteintech, Chicago, IL, United States); goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody (ZSGB Biotechnology, Beijing, China). Lipopolysaccharides (LPS, E. coli L2630) and 3-MA (M9281) were from Sigma-Aldrich (St. Louis, MO, United States), and rapamycin was from Beyotime Biotechnology (Wuhan, Hubei, China). Caspase-1 inhibitor Z-YVAD-FMK was purchased from Biovision (San Francisco, CA, United States). The ELISA kit for mouse IL-1β (88-7013) was purchased from eBioscience Technology (San Diego, CA, United States). Reagents and apparatus used in immunoblotting assays were obtained from Bio-Rad (Hercules, CA, United States).