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Supporting Information Supporting Information Jensen et al. 10.1073/pnas.1316700111 SI Materials and Methods Western Blot Analysis. For all Western blot analyses, proteins were Cloning of Bcl2L13 and Mutagenesis of Bcl2L13. Full-length Bcl2-like separated by 4–12% SDS/PAGE (Life Technologies), transferred 13 (Bcl2L13) was cloned into pCDNA3-V5-His (Invitrogen) to Hybond PVDF membranes (Amersham), blocked with 5% using unique EcoRV/XhoI restriction sites and into the CSII- (wt/vol) milk in PBS with 0.1% Tween 20 (PBS/Tween) for 1 h, CMV-MCS-IRES2-Venus lentiviral vector via NheI/AgeI re- washed with PBS/Tween, and incubated with the following an- striction sites. To delete the BHNo domain (amino acids 207–459, tibodies: anti-actin (Santa Cruz), anti-Bax (Millipore), anti- – base pairs 621–1377) of Bcl2L13, mutagenesis PCR was em- Bcl2L13 (Proteintech), anti cleaved caspase-3 (Cell Signaling), – – ployed using primers ATACTGCTGTTTGGAGGGGCTGCT- anti cleaved caspase-7 (Cell Signaling), anti cleaved caspase-9 GCTGTTGCC and AAGACTAAACACACAGTGCCCCAGC- (Cell Signaling), anti-COXIV (Cell Signaling), anti-COX17 (Santa Cruz), anti–Bcl-2 (Millipore), anti–Bcl-x (Cell Signaling), CACCTTG that amplified a mutant Bcl2L13 lacking the BHNo L anti-Bak (Cell Signaling), anti-Hsp70 (BD Pharmingen), anti- domain (ΔBHNo). The mutant PCR product was ligated back LASS2 (CerS2; Abnova), anti-LASS6 (CerS6; Abnova), anti- together and was transformed. FLAG (Sigma), anti-HA (Santa Cruz), and anti-V5 (Invitrogen). Lentiviral Production and Infection. 293FT cells were plated in a The blots were washed with PBS/Tween and subsequently were 10-cm dish at 3 × 106 cells per plate. After 24 h, cells were trans- incubated with goat anti-rabbit IgG or with goat anti-mouse fected with 3 μg of target lentiviral construct, 1.25 μg of pMD2.G antibodies (Santa Cruz) in 5% (wt/vol) milk PBS/Tween. After washing with PBS/Tween, the blots were developed with ECL (envelope), and 3.75 μg psPAX2 (HIV-Gag-Pol-Rev) and were Plus (GE Healthcare) following the manufacturer’s protocol. incubated overnight. The next day, fresh medium was added, and 48 h posttransfection medium was harvested, supplemented with RT-Quantitative PCR. To analyze Bcl2L13 expression levels in μ Polybrene (Sigma-Aldrich) (8 g/mL), and filtered through a glioblastoma (GBM) tumor samples, RT-quantitative PCR (RT- 0.45-μM syringe filter. Virus-containing medium was stored at qPCR) was performed using cDNA isolated from primary GBM −80 °C until infection. For lentiviral transduction, cells at 50% tumor samples acquired in compliance with the Northwestern confluence were incubated overnight with 3 mL of virus-containing Memorial Hospital Institutional Review Board from patients medium before changing to virus-free medium. At 48 h postin- having undergone surgery at Northwestern Memorial Hospital. fection cells were sorted by FACS to enrich Venus-positive cells Total RNA was extracted from snap-frozen specimens by dis- (for the CSII construct) or were puromycin-selected (for pGIPZ sociation (mortar and pestle grinding) followed by resuspension shRNA constructs). in RLT buffer (Qiagen). Total RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer’s protocol. Transient Overexpression and siRNA Transfection. Subconfluent cDNA was synthesized via M-MLV Reverse Transcriptase re- glioma cells were transfected with a nontargeting siRNA control, actions (Promega), according to the manufacturer’sprotocol, with small, interfering ceramide synthase 2 (siCerS2), or with using 500 ng of total RNA as template. RT-qPCR was performed small, interfering ceramide synthase 6 (siCerS6) siRNA pools using TaqMan probes (Life Technologies) specific to Bcl2L13 (Dharmacon) at 50 nM using Oligofectamine (Invitrogen) according (assay ID: Hs00209787_m1) and 18S (assay ID: Hs99999901_s1; to the manufacturer’s protocol. Apoptosis assays (see below) were used as an endogenous control), on a 7500 Real Time PCR performed 48 h after transfection. Transient transfections of CerS System (Applied Biosystems). Expression of Bcl2L13 in GBM cDNAs were performed with 140–400 ng/cm2 (plasmid DNA/ tumors was compared with a normal brain reference pool con- surface area) cDNA using Lipofectamine 2000 (Life Technologies) sisting of 23 individual brain samples (FirstChoice Human Brain according to the manufacturer’s protocol. Apoptosis assays (as Reference, catalog no. AM6050; Life Technologies). Results were ΔΔ described below) were performed 24–48 h after transfection. For analyzed and quantified using the Ct method. transfection of the ΔBHNo mutant Bcl2L13 (ΔBHNoV5), 400 ng/cm2 of plasmid DNA was transfected, compared with 200 ng/cm2 Deconvolution Immunofluorescence Microscopy and Immunohisto- chemistry. For immunofluorescence studies, LN235 and SF767 for full-length Bcl2L13, to adjust for differences in expression effi- cells were grown on poly-D-lysine–coated slides or in eight-well ciencies and to allow equal expression levels of both constructs. chambers (LabTek) and were fixed with 4% paraformaldehyde for 10 min at 37 °C. After fixation, the slides were washed three times Coimmunoprecipitations. Coimmunoprecipitations (co-IP) were performed using the Dynabead System (Life Technologies) accord- with PBS, permeabilized with 0.5% Triton X-100, and incubated in blocking solution [0.1% Triton, 5% (wt/vol) BSA] for 1 h ing to the manufacturer’s protocol, with slight modifications. at 37 °C. Slides were incubated overnight at 4 °C with the fol- Briefly, SF767 cells were harvested 24 h after transfection and lowing antibodies: anti-Bcl2L13 (Proteintech), anti-cytochrome c subsequently were lysed in co-IP lysis buffer [0.4% Nonidet P-40, (BD Pharmingen), anti-HA (Santa Cruz), and anti-V5 (In- 142 mM KCl, 20 mM Hepes, 1 mM EDTA, with protease in- vitrogen). After incubation with Alexa Fluor 488 or Alexa Fluor hibitor mixture (Calbiochem)]. Dynabeads were equilibrated, 594 secondary antibodies (Life Technologies), images were conjugated to anti-Flag antibody (Sigma) or mouse IgG (Santa generated and deconvolved on a Zeiss UV LSM510 confocal Cruz) by mixing end-over-end, and washed once with PBS. Then microscope. – 0.5 2 mg of protein lysate was added to the Dynabeads-antibody For immunohistochemistry, brain and tumor tissue was de- conjugate and was incubated for 30 min at room temperature with paraffinized in xylene, rehydrated with a series of decreasing end-over-end mixing. The protein-bound Dynabeads then were EtOH to distilled water concentrations, and placed into citrate washed four times with PBS, and protein was removed from the antigen retrieval solution (10 mM sodium citrate, 0.1% Tween Dynabeads by resuspending the beads in 20 μL of NuPAGE LDS 20, pH 6). Antigen was retrieved at 120 °C for 3 min. Slides were β Sample Buffer (Life Technologies) containing -mercaptoethanol cooled, washed in PBS, and treated with 1% H2O2 for 20 min to and by heating at 70 °C for 10 min. remove endogenous peroxidase activity. Slides were washed Jensen et al. www.pnas.org/cgi/content/short/1316700111 1of9 again with PBS, blocked with normal goat serum for 1 h at room were postfixed in 2% gluteraldehyde/TBS for 5 min. Finally, temperature, and incubated overnight with a polyclonal anti- grids were counterstained for 5 min in 3% uranyl acetate and Bcl2L13 antibody (Proteintech). Slides were processed further and lead citrate. Sections were imaged on an FEI Tecnai Spirit developed using the ABC Vectastain kit (Vector Labs) according to transmission electron microscope. the manufacturer’s protocol. After processing, slides were dehy- drated with a series of EtOH washes, coverslipped, and then ana- Bax Mitochondrial Insertion and Oligomerization Assays. To measure lyzed by bright-field microcopy. Bax insertion into the mitochondrial membrane, cells were treated with STS (100 nM) or curcumin (20 μM) for the indicated Quantification of Apoptosis. Apoptosis was induced using the pan- periods of time, washed in PBS, and permeabilized by adding specific kinase inhibitor staurosporine (STS, 100–500 nM; Sigma), 1 mL of 0.02% digitonin in isotonic buffer A (10 mM Hepes, 150 curcumin (8–50 μM), and the receptor tyrosine kinase inhibitors mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, pH 7.4) followed by erlotinib (5 μM; Sigma), SU11274 (5 μM; Sigma), imatinib (5 μM; mixing end-over-end for 2 min at room temperature. Cells then Selleck Chemicals), AG1024 (5 μM; Sigma), KI8751 (2 μM; Sigma) were centrifuged at 15,000 × g for 10 min at 4 °C. The resulting for indicated periods of time. The degree of apoptosis was de- supernatant represents the cytosolic fraction, and the pellet termined by Western blotting for cleaved caspases-3, -7, and -9, by a contained organelles and organelle-bound proteins (including fluorometric cleaved caspase-3/7 assay (Caspase-3/CPP32 Fluoro- Bax in apoptotic cells). Membrane-bound proteins were ex- metric Assay Kit; Biovision), by a mitochondrial membrane de- tracted from the organelles by resuspending the pellet in 100 μL polarization assay (FlowCellect MitoDamage Kit; Millipore), or by of 2% CHAPS in buffer A, incubated on ice for 60 min with FACS-based Annexin V staining (Biovision), all used according to occasional vortexing, and then centrifuged at 15,000 × g for the manufacturer’s instructions. 10 min. The resulting membrane soluble fraction contained mem- brane-associated proteins (including Bax). The amount of Bax in Cytochrome c Release Assay. SF767 cells were exposed to erlotinib, the cytosolic and membrane-soluble fraction was determined by SU11274, imatinib, AG1024 (at 5 μM each) and to KI8751 anti-Bax Western blotting. (2 μM) for 24 h, STS (100 nM) for 4 h, or curcumin (200 μM) for To measure Bax oligomerization in response to curcumin- or 2 h. Cytoplasmic and heavy membrane fractions were separated STS-induced apoptosis, cells were washed and harvested in PBS as previously described (1), and the amount of cytochrome c and permeabilized with 0.02% digitonin in isotonic buffer A, and released into the cytosol was quantified using the Quantikine membrane-bound proteins were extracted with CHAPS as de- Cytochrome c ELISA kit (R&D Systems) according to the scribed above.
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