Mntbap

Unilateral ureteral obstruction (UUO) was induced as described previously [12 (link), 31 (link), 34 (link)]. Briefly, the left ureter was exposed and subsequently ligated with 6.0 silk through a small abdominal incision under the anesthesia with 2.0% isoflurane. The abdomen was closed in two layers. All mice received analgesia (subcutaneous injection of 50 μg/kg buprenorphine (Temgesic, Schering-Plough)) after the surgery. Following the surgery, the UUO mice were treated with Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) (Catalog No.: sc-221954A, Santa Cruz, Dallas, TA, USA), at a dose of 10mg/kg/day in water by intraperitoneal injection. The control mice received sham operation and were treated with the same volume of water. All mice (N = 6 per group) were sacrificed 7 days after operation and the kidney tissues were harvested for further analysis.

Phorbol-12-myristate-13-acetate (PMA), MSU, ATP, nigericin, poly A:T, Pam3CSK4, A9C, DIDS, and IAA94 were purchased from Sigma. MitoSox, MitoTracker Red, DAPI, MQAE, Lipofectamine RNAiMAX, Lipofectamine 2000, and M-MLV were from Invitrogen. Ultrapure LPS was obtained from Invivogen. SYRB Green was from TransGen Biotech. MnTBAP was from Santa Cruz. Salmonella is a gift from R.V. Bruggen. Anti-human cleaved IL-1β (2021, 1:1000 dilution) and anti-human caspase-1(2225, 1:1000 dilution) antibodies were purchased from Cell Signalling. Anti-human pro-IL-1β (60136-1-lg, 1:1000 dilution) anti-CLIC1(14545-1-AP, 1:500 dilution), anti-GAPDH (10494-1-AP, 1:1000 dilution), anti-VDAC1 (66345-1-lg, 1:1000 dilution), and anti-ATP1A1 (55187-1-AP, 1:1000 dilution) antibodies were from Proteintech. Anti-CLIC4 (SC-135739, 1:1000 dilution), anti-NEK7 (SC-50756, 1:500 dilution), and anti-ASC (rabbit, SC-22514, 1:400 dilution) antibodies were from Santa Cruz. Anti-ASC (mouse, 04–147, 1:1000 dilution) antibody was from Merck Millipore. Anti-CLIC5 (ACL-025, 1:250 dilution) antibody was from Alomone labs. Anti-mouse IL-1β (AF-401-NA, 1:1000 dilution) antibody was from R&D. Anti-mouse caspase-1 (p20) (AG-20B-0042, 1:1000 dilution) and anti-NLRP3 (AG-20B-0014, 1:1000 dilution) antibodies were from Adipogen. Anti-β-actin (P30002, 1:1000 dilution) antibody was from Abmart.

Apilimod (No. S6414) was bought from Selleck. Nigericin, MSU, and poly (A/T) were purchased from Sigma-Aldrich. Ultrapure LPS, MitoSOX, MitoTracker, DAPI, Lysotracker Green, MitoTracker Deep Red, MitoTracker Green were acquired from Invitrogen. MnTBAP was bought from Santa Cruz Biotechnology. The C3 toxin was a gift from Dr. Tengchuan Jin (University of Science and Technology of China, Hefei, China). CY-09 was synthesized from Dr. Xianming Deng (Xiamen University, Xiamen, China). MCC950 was bought from Selleck. Fluo-4, AM(HY-101896), ML-SI3 were purchased from MedChemExpress. BAPTA-AM(A1076) was bought from Sigma-Aldrich. Protein G agarose (16–266) was bought from Millipore. Anti-Flag (F2555) antibodies were from Sigma-Aldrich. The anti-mouse NLRP3 (AG-20B-0014) and the anti-mouse caspase-1 (AG-20B-0042) antibodies was from AdipoGen. The anti-mouse IL-1β antibody (AF-401-NA) was obtained from R&D Systems. The anti-β-actin antibody (66009-1-Ig) was from Proteintech Group. The anti-mouse ASC antibody (67824S) was from Cell Signaling Technology. The anti-mouse NEK7 antibody (ab133514) was from Abcam.

For Figures 3b and 5d, a modified, non-radioactive version of our chromium-based assay was conducted using the CytoTox-ONE Homogenous Membrane Integrity assay (Promega), which measures LDH release as a measure of cell death. Assay setup was similar to the chromium assay, excluding the chromium loading step and a 50:1 E:T ratio of PBMCs to tumor cells (because of the higher sensitivity of this assay). After 4 h incubation of cells at 37 °C, supernatant (50 μL) was collected and mixed with 50 μL CytoTox-ONE reagent. Luminescence was measured using a BMG FLUOstar OPTIMA (BMG Labtech, Cary, NC, USA) and percent lysis solely due to ADCC was calculated by: %specific lysis (PBMC+antibody)-%specific lysis (PBMC alone). For reversal experiments, antioxidant SOD mimetic MnTBAP (Santa Cruz Biotechnology, Dallas, TX, USA), or caspase inhibitor qVD-OPh (EMD Millipore, Billerica, MA, USA), alone and in combination, were added during antibody incubation. For inhibition of NF-κB activation, NRAGE peptide (described above) was added with 6 μM EndoPorter delivery reagent (GeneTools, LLC, Philomath, OR, USA) to facilitate intracellular transport.

B5G9 with a purity of 98% was synthesized as described previously [24 (link)]. Hoechst 33342, H₂DCFDA, MitoTracker® Red CMXRos, MitoSOX Red, a BCA protein assay kit and a Dead Cell Apoptosis Kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Necrostatin-1 was obtained from Selleck Chemicals (Houston, TX, USA). MnTBAP was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). An antibody against cytochrome c was obtained from Epitomics (Burlingame, CA, USA). Antibodies against caspase-3, caspase-9, cleaved-caspase-3, cleaved-caspase-9, PARP and β-actin were obtained from Cell Signaling (Beverly, MA, USA). Other reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

Staining of 5 µM sections of the frozen brains with dihydroethidium (DHE) and DAPI was performed in the dark to determine superoxide generation in the brain sections. Incubation of additional sections with 100 μM of the SODm, MnTBAP (Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States) for 10 min prior to DHE was applied in matched control experiments to confirm the specificity of the fluorescence and prove that signals are derived from superoxide. The slides were rinsed extensively with PBS, mounted in antifade mounting media Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL, United States), cover slipped, then different fields were captured at ×40 using a confocal microscope (Olympus FV3000 spectral confocal microscope, Tokyo, Japan) with excitation wavelength at 495 nm and emission at 515 nm, and the fluorescence intensity was analyzed using the microscope provided software.

Samples were sectioned in a cryostat (8 μm) and incubated with 150 μM of MnTBAP (Santa Cruz Biotechnology) for 1 h at RT. The samples were then washed with PBS and incubated with 1 μM of dihydroethidium (DHE, Sigma-Aldrich) for 30 min at 37 °C. Sections were washed again and mounted with ProLong mounting media containing DAPI (Invitrogen). Photographs were taken with a fluorescence microscope (Axioimager.D1 Zeiss) and analyzed by ImageJ software.