Ca 074me

TMAO was purchased from Sigma, inhibitors included CA-074Me (Selleck, S7420) and MCC950 (MCE, HY-12815A). Immunoblotting was performed using primary antibodies Cathepsin B (CST, #31718), NLRP3 (CST, #15101), Cleaved IL-1β (Abcam, ab9722), Cleaved Caspase-1 (CST, #2225), eNOS (CST, #32027), p-eNOS (CST, #9570), ZO-2 (BOSTER, BA 2861), OCLN (BOSTER, BM 4832).

Mouse peritoneal macrophages were isolated and cultured as described previously (24 (link)). Briefly, mice (8 weeks old) were injected with 3 ml of sterile 3% thioglycollate broth intraperitoneally to elicit the formation of peritoneal macrophages. After 72 h, cells were collected via lavage of the peritoneal cavity with 10 ml of RPMI 1640 medium (GIBCO). Then, cells were centrifuged at 800 rpm and were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (GIBCO) and 1% penicillin–streptomycin (Thermo Fisher). Peritoneal macrophages (10⁶ cells per well) were plated in 12-well plates and then primed with ultra-pure LPS (100 ng/ml) for 4 h, and then stimulated with SiO₂ crystal (20 μg/ml), APC-connected silica crystals (20 μg/ml), or liposome-coated silica crystals (20 μg/ml) for 6 h. To induce lysosomal disruption, peritoneal macrophages were primed with ultra-pure LPS (1 ng/ml) for 4 h and then were stimulated with Leu-Leu-OMe·HCl (1,000 μM, Chem-Impex International) for 5 h. To inhibit the activity of cathepsin B, peritoneal macrophages were primed with 100 μM CA-074Me (Selleck) overnight before ultra-pure LPS priming.

Monocytes were directly stimulated after isolation in either standard RPMI1640 cell culture medium (high [P_i], 5.6 mM Na₂HPO₄, Gibco, Lifetechnologies) or customized RPMI1640 containing 1 mM Na₂HPO₄ (low [P_i]) supplemented with 10% FBS (Gibco, Lifetechnologies). 3 × 10⁵ monocytes were seeded in 96-well plates and 100 ng/ml LPS (Invivogen) was used for monocyte “priming”. Inhibitors were pre-incubated for 30–60 min prior to stimulation.
CaSR (established in our group) and NLRP3-deficient (Invivogen) THP-1 cells were cultured in RPMI1640/10% FBS/1% penicillin and streptomycin (pen/strep) and selection antibiotics as indicated in the data sheet. Assays were performed in 24-well plates. 5 × 10⁵ cells/well were plated for differentiation in 50 ng/ml PMA (Tocris)-containing medium for 2 days before LPS-priming (100 ng/ml) and stimulation.
The following reagents and inhibitors were used for several cell culture experiments. Na₂HPO₄, BaCl₂, fetuin-A from FBS were purchased from Merck, CaCl₂, sodium phosphonoformate tribasic hexahydrate, from Sigma, YM254890 from Wako chemicals, Calhex231, NPS2143, Latrunculin A, Cytochalasin D from Tocris, Latrunculin B, PAF C-16, LLOMe from CaymanChemicals, MgSO₄ from AppliChem, ATP from Roche, DMSO from Serva, N-fMLP from abcam, and CA-074-Me from Selleck-Chem.

CA074me (Selleckchem) and PADK (Bachem) treatments were performed at the indicated final concentrations with DMSO as vehicle. For PFF experiments in Figs. 2 and 3, a single drug treatment was performed simultaneous with PFF administration, after which media was refreshed every 5–7 days. For lysosomal assays in Fig. 3, drug was administered 5 days prior to the assay unless otherwise specified.
For PFF treatments on RPE1 cells, 50,000 cells were plated on 12-well plates. After 24 hours, PFF was added and cells were allowed to continue growing for 48 hours before cells were washed with PBS and dissociated with trypsin to remove non-internalized PFFs before being lysed in RIPA buffer.
For PFF high-content imaging assays with PFF, neurons were treated with PFF after 2 weeks of differentiation in 96-well plates. After treatment, media was refreshed every 5–7 days normally. At completion of treatment, cells were washed with PBS and fixed with 4% paraformaldehyde.

In some ConA-treated mice, 5 mg/kg CA-074 Me (a specific inhibitor of cathepsin B) (Selleck Chemicals, diluted in 5% DMSO containing PBS) or vehicle was administrated by i.p injections daily and before sacrifice.

Doxorubicin (10 mM in water), topotecan (10 mM in water), naringenin (100 mM in DMSO), chloroquine (100 mM in water), and 5-fluorouracil (50 mM in water) were purchased from Sigma Aldrich (Taufkirchen, Germany). Vincristine (100 mM in DMSO) was purchased from Adipogen (San Diego, CA, USA). Tetrandrine (10 mM in DMSO) was purchased from Sigma Aldrich and purified by the group of Prof. Franz Bracher (Ludwig-Maximilians University, Munich). CA-074Me (50 mM in DMSO) was purchased from Selleckchem (Houston, TX, USA).