Highly purified human recombinant TNF was provided by BASF Bioresearch (Ludwigshafen, Germany). Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) was from Bachem (Bubendorf, Switzerland). TPCK, marimastat, benzyloxycarbonyl-Phe-Ala-fluoromethylketone (zFA-fmk) and trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), were purchased from Sigma (Deisenhofen, Germany), necrostatin-1, TAPI-1, GM6001, 5-[5-(2-nitrophenyl)furfurylidine]-1,3-diphenyl-2-thiobarbituric acid (Ucf-101), benzyloxycarbonyl-Phe-Phe-fluoromethylketone (zFF-fmk) and LDN57444 from Merck Millipore (Darmstadt, Germany), and N-[L-3-trans-(propylcarbamoyl)-oxirane-2-carbonyl]-L-Ile-L-Pro methyl ester (CA-074 Me) from Biomol (Hamburg, Germany). Carboxyfluorescein-labeled phenylalanine chloromethyl ketone (FAM-FFCK) was from Immuno Chemistry Technologies (Bloomington, MN, USA). Staurosporine was obtained from Selleckchem (Munich, Germany), Ubiquitin vinyl methyl ester, HA-tag (HaUbVME) from Enzo Life Sciences (Lausen, Switzerland).
L929Ts is a TRAIL-sensitive L929 subline derived in our laboratory
. NIH3T3 cells naturally expressing RIPK3 and therefore sensitive to necroptosis have been previously described
[15, 50, 51]. Jurkat and HT-29 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Jurkat I42 cells were a kind gift from Francis Ka-Ming Chan (Worcester, MA, USA). Immortalized MEF deficient for HtrA2/Omi
 and their WT counterparts were originally generated by Julian Downward (London, U. K.) and kindly provided by Thomas Langer (Cologne, Germany). Cells were cultivated in DMEM (NIH3T3, MEF), or a mixture of Click’s/RPMI 1640 medium (all other cell lines) supplemented with 10% v/v fetal calf serum and 2 mM glutamine at 37°C in a humidified incubator containing 5% w/v CO2. Media were additionally supplemented with 1 mM sodium pyruvate (HT-29) and 50 μg/ml each of streptomycin and penicillin. Murine podocytes (a kind gift from K. H. Endlich, Greifswald) were cultured as described
. For differentiation, podocytes were cultured for 14 days under non-permissive conditions (37°C, 7.4% w/v CO2, RPMI 1640 supplemented with 10% v/v fetal calf serum, 10 mM N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin).
Flow cytometric analysis of membrane integrity
Cells were seeded in twelve-well plates at 5 x 104 cells/well. Following treatment, both detached and adherent cells were collected by centrifugation. The cells were resuspended in PBS/5 mM EDTA containing 2 μg/ml propidium iodide (PI), and the red fluorescence was measured on a FACSCalibur flow cytometer (Becton Dickinson).
p values were calculated using Student’s t-test. Statistical significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001.
For documentation of cell morphology, images from unfixed cells were obtained using an Axiovert 10 microscope (Zeiss, Oberkochen, Germany) and a DS-5M-L1 digital sight camera system (Nikon, Düsseldorf, Germany).
2D gel electrophoresis, image analysis and spot picking
The two-dimensional gel electrophoresis was essentially performed as described before
. After harvesting, cells were lysed on ice for 10 min in TNE buffer (50 mM Tris pH 8.0, 1% v/v NP40, 2 mM EDTA) containing 10 μg/ml protease inhibitor cocktail (Roche, Mannheim, Germany). For protein precipitation, trichloroacetic acid (TCA) was added to the protein lysate to a final concentration of 10% v/v . The mixture was incubated for 30 min on ice and centrifuged at 10,000 × g at 4°C for 20 min. The supernatant was removed, ice-cold acetone was added to wash the pellet and the sample was centrifuged as above. After removal of the supernatant, the pellet was air dried and resuspended in lysis buffer (pH 8.5) containing 7 M urea, 2 M thiourea, 30 mM Tris, 4% w/v CHAPS. The supernatant containing the solubilized proteins was recovered after centrifugation for 20 min at 20,000 × g at 4°C. A total amount of 250 μg of protein was mixed with rehydration buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 2% v/v immobilized pH gradient (IPG) buffer pH 3–11 and 2% w/v DTT) and applied by cup-loading onto 24 cm non-linear pH 3–11 IPG gel strips for isoelectric focusing (IEF). The second dimension was performed on 26 × 20 cm large 12.5% w/v gels after reduction and alkylation using the Ettan DALTsix large vertical electrophoresis system from GE Healthcare (Munich, Germany). The gels were removed from the glass plates, mounted on a non-backed gel frame, and scanned on a Typhoon Trio imager (GE Healthcare) at green fluorescence. Subsequently, the gels were stained overnight with Flamingo Pink (Bio-Rad, Munich, Germany), and scanned again at red fluorescence. The obtained images were analyzed using Image Master 6.0 (GE Healthcare). Selected spots were picked with a 2 mm picking head. The picked gels were again scanned to verify the correct location of the punched spots.
In-gel tryptic digestion and mass spectrometry
The punched gel spots were sequentially washed with water, with 50 mM ammonium bicarbonate (ABC) in 50% v/v MeOH, with 70% v/v acetonitrile (ACN), and dehydrated in pure ACN. ACN was evaporated in a SpeedVac centrifuge (ThermoFisher Scientific, Dreieich, Germany), and the dry gel pieces were subjected to in-gel digestion with 100 ng porcine sequencing-grade trypsin (Serva, Heidelberg, Germany) in 25 mM ABC at 37°C overnight. For peptide extraction, 20 μl of 0.1% v/v trifluoroacetic acid (TFA) in ACN was added and the samples were sonicated for 15 min. The supernatants were removed and the gel spots were again incubated with 20 μl of 0.1% v/v TFA in ACN for 10 min. The supernatants of both steps were combined, dried in a SpeedVac centrifuge, redissolved in 0.8 μl MALDI matrix solution (3.2 mg/ml α-cyanohydroxycinnamic acid (Sigma) in 65% v/v ACN/0.1% v/v TFA), spotted onto 384-well stainless steel MALDI plates and air-dried. Spectra were acquired on an AB SCIEX MALDI-TOF/TOF 5800 (AB SCIEX, Darmstadt, Germany) mass spectrometer in positive ion mode. For MS measurements, 2000 shots were accumulated in the mass range of 800–4000 m/z. Default calibration was performed using the 4700 Proteomics Analyzer Standards Kit, while MS measurements were calibrated internally using trypsin and contaminant peaks (842.509, 2211.105, 2225.120 and 2807.315 Da). Precursor selection for MS/MS analysis was achieved using the 4000 Series Explorer Software (AB SCIEX) with acquisition of the 20 most intense precursors (S/N > 20), beginning with the strongest first. All MS/MS spectra were acquired with 1 KV collision energy at ambient air (CID medium: 1.25 x 10–6 Torr) using 3000 laser shots. For peptide identification, MALDI-TOF/TOF MS/MS raw files were searched using ABSciex GPS software (Version 3.6, build 332) with the following pre-filter settings: only peaks within a mass range from 60 Da to the precursor mass minus 35 Da and S/N ratio above 10 were used. Spectra were searched with Mascot (version 2.2.04, Matrix Science, London, U.K) against the Swissprot database using Mus musculus as a taxonomy filter (15 Feb 2011, 16345 sequences) and the following parameters: precursor tolerance, 50 ppm; MSMS tol, 0.3 Da; max missed cleavages 2. Oxidation (M) was set as a variable modification, while carbamidiomethylation (C) was set as a fixed modification. Proteins were considered identified when either 2 peptides were identified with a confidence interval ≥ 99% (p < 0.01) or 3 peptides ≥ 95% (p < 0.05).
The validated siRNA specific for human HtrA2/Omi (ID # s654), the predesigned siRNAs specific for murine HtrA2/Omi (ID # s82292, s82292) murine UCH-L1 (ID # s75710), murine RIPK3 (ID # s80755) as well as the negative control siRNA (ID # AM4611) were obtained from Life Technologies, Darmstadt, Germany. L929Ts cells were transfected with 150 pmol siRNA by Amaxa nucleofection (Lonza, Cologne, Germany), using solution V and program T-20. Jurkat I42 cells were transfected with 30 pmol siRNA and HiPerFect transfection reagent (Qiagen, Hilden, Germany).
Measurement of intracellular ATP levels
The intracellular ATP content of cells was determined with the Cell Titer Glo Assay Kit (Promega, Mannheim, Germany) following the instructions of the manufacturer.
Unless otherwise indicated, cells were harvested after treatment and lysed at 4°C in TNE buffer containing 150 mM NaCl, 10 μg/ml protease inhibitor cocktail, 1 mM sodium orthovanadate and 5 mM NaF. Identical amounts of cell protein per lane were resolved by electrophoresis on SDS polyacrylamide gels. After electrophoretic transfer to nitrocellulose, reactive proteins were detected using antisera specific for actin (sc-1615, Santa Cruz, Heidelberg, Germany; A1978, Sigma), HtrA2/Omi (ab32092, Abcam, Cambridge, UK), UCH-L1 (rat monoclonal, clone U104, generated as outlined below, or rabbit polyclonal, CL95101, Cedarlane, Burlington, Ontario, Canada), HA (1187423, Roche), PARP-1 (9542, Cell Signaling, Danvers, MA, USA) and the ECL detection kit (GE Healthcare). Equal loading as well as efficiency of transfer was routinely verified for all Western blots by Ponceau S staining, and by reprobing the membranes for actin.
Generation of monoclonal UCH-L1 antibodies
Wistar rats were initially immunized intraperitoneally (i.p.) with 100 μg of purified UCH-L1 (kindly provided by Gregory A. Petsko, Waltham, MA, USA) in 60 μl phosphate buffer saline (PBS) emulsified with 40 μl of Gerbu adjuvant MM (Gerbu Biotechnik, Heidelberg, Germany). The rats were boosted i.p. on days 14 and 21 with 50 μg of purified protein emulsified with 20% v/v of the adjuvant. The last two doses (50 μg UCH-L1 in PBS) were administered on days 28 and 29 without adjuvant, while the fusion was done on day 30. Spleen cells from immunized animals were collected and fused with Ag8.653 myeloma cells using polyethylene glycol 1500 (Roche). The fused cells were cultured in selection medium (HAT, Sigma) for 10 days and screened by ELISA for anti-UCH-L1 antibodies. Hybridoma clones producing anti-UCH-L1 monoclonal antibodies (mAbs) were then cultivated in serum-free medium and the mAbs were purified using protein G affinity chromatography (GE Healthcare). The isotype of the anti-UCH-L (U104) clone (IgG1, λ) was determined by using ELISA rat mAb isotyping kit (ThermoFisher).
Cellular lysates were precleared with GammaBind G-sepharose (GE Healthcare) and immunoprecipitation was performed over night on ice using anti-ubiquitin IgG1 monoclonal antibody (MAB1510, Merck Millipore, 1:100 dilution). After collection of the immunecomplexes with GammaBind G-sepharose and three washing steps in lysis buffer, the immunoprecipitated proteins were analyzed by SDS-PAGE and Western blot.
Generation of stably transfected podocytes with inducible overexpression or downregulation of UCH-L1
For inducible overexpression of UCH-L1, the Retro-X Tet-On Advanced Inducible Expression System (Clontech, Mountain View, CA, USA) was used according to the manufacturers’ instructions. Briefly, wildtype murine UCH-L1 was amplified by polymerase chain reaction from murine podocytes using the following primers: mUCHL1-pRetro-fw 5′CTAGGCGGCCGCGCCACCATGCAGCTGAAGCCGATGGA′3; mUCHL1-pRetro-rev 5′CTAGACGCGTTTAAGCTGCTTTGCAGAGAG′3 and subsequently cloned into the multiple cloning site of the pRetroX-Tight-Pur vector using NotI and MluI (ThermoFisher). The sequence of UCH-L1 was verified by sequencing (Eurofins MWG Operon, Ebersberg, Germany). For virus production, phoenix ecotropic packaging cells were transfected using DNA/CaCl2 precipitation with the pRetroX-Tet-On Advanced vector, with the pRetroX-Tight-Pur-UCH-L1 vector or the pRetroX-TightPur empty vector as a control, respectively. The virus-containing supernatant of the pRetroX-Tet-On transfected phoenix cells was transferred to a 10 cm plate containing podocyte target cells at around 50% to 60% confluence; the infection steps were repeated twice. Selection for integration of the pRetroX-Tet-On Advanced expression plasmid was performed with G418 (500 μg/ml, Life Technologies) for 7 days. Afterwards, the virus-containing supernatant of the pRetroX-Tight-Pur-UCH-L1 transfected phoenix cells was transferred to the pRetroX-Tet-On Advanced transduced podocyte target cells; the infection steps were again repeated twice. Selection for integration of the pRetroX-Tight-Pur-UCH-L1 plasmid was performed with puromycin (1.5 μg/ml, Sigma). For negative control experiments, the pRetroX-Tight-Pur vector was transduced without insert (tet-) into the pRetroX-Tet-On Advanced expressing podocytes. For induction of UCH-L1 overexpression, UCH-L1 tet-on or tet- podocytes were cultured in the presence of tetracycline free medium (PAN-Biotech, Aidenbach, Germany) supplemented with 20 ng/ml doxycycline or without doxycycline for control. For stable knockdown experiments, shRNA627 to murine UCH-L1 or scrambled shRNA for control was overexpressed in podocytes as described before
Analysis of caspase activity, cell death, and cellular and nuclear morphology in podocytes
105 differentiated UCH-L1 tet-on or tet- podocytes were plated in 6-well plates in tetracycline-free RPMI 1640 medium (Life Technologies) supplemented with 10% v/v fetal calf serum, 10 mM N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin. UCH-L1 overexpression was induced with 20 ng/ml doxycycline for 72 hours or not. For measurements of caspase activity, cells were collected and lysed in a buffer containing 10 mM Hepes pH 7.4, 142 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.2% v/v NP40, 1 mM DTT and 2 mM Pefabloc (Roche). To generate positive controls, 20 μg of cells lysate were equilibrated for 1 h at 30°C after the addition of 1 mM dATP and 10 μM cytochrome c to permit activation of caspases. Subsequently, 100 μl of caspase buffer (20 mM Pipes, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% w/v CHAPS, 10% w/v sucrose, pH 7.2) containing 100 μM zDEVD-afc (benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-7-aminotrifluoromethylcoumarin, Merck Millipore) or zIETD-afc benzyloxycarbonyl-Ile-Glu(OMe)-Thr-DL-Asp(OMe)-7-aminotrifluoromethylcoumarin (Merck Millipore) were added to 10 μl of cytosolic extract (10 μg protein) and incubated at 37°C. The release of afc was measured as emission at 505 nm upon excitation at 405 nm using an Infinite M200 fluorimeter equipped with a thermostated plate reader (Tecan, Crailsheim, Germany). For measurements of podocyte death, viable and dead cells were detached with trypsin and counted in a Neubauer chamber after 0.1% w/v trypan blue (Life Technologies) staining. The percentage of dead cells was calculated and plotted as mean +/- SEM, n = 12 per condition. To analyze cellular and nuclear morphology, cells were stained with Hoechst dye (10 μg/ml, Life Technologies) for 5 min and DNA condensation in UCH-L1 tet-on podocytes with or without induced UCH-L1 overexpression for 72 hours was evaluated under an Axio Observer A1 microscope (Zeiss) using the axiovision software (Zeiss).
Analysis of TNF-induced cell death in podocytes
Differentiated sh627 and scrambled shRNA control podocytes were plated at a density of 104 cells per 6-well plate. After 48 hours, cells were treated with 100 ng/ml murine TNF (PeproTech, Hamburg, Germany) with addition of 50 μM zVAD-fmk or vehicle (ethanol) as control for 3 hours. Cells were detached with trypsin and the amount of dead and living cells was counted in a Neubauer chamber following staining with 0.1% w/v trypan blue. The percentage of dead cells was calculated and plotted as mean +/- SEM, n = 12 per condition.