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. Then membrane-bound proteins (including Bax) manufacturer’s protocol. The truncated BH3 interacting domain were cross-linked with 1 mM ethylene glycol bis[succinimidyl- death agonist (tBid)-induced cytochrome c release from isolated succinate] (Thermo Fisher) for 30 min by end-over-end rocking mitochondria was measured as previously described (1) with at room temperature. The cross-linking reaction was quenched modifications. tBid in dilution buffer [20 mM Hepes (pH 7.4), 10 by adding 0.1 volume of 2-M Tris·HCl, pH 7.4, followed by in- mM KCl, 20 mM MgCl2, 1 mM EDTA] was incubated with 50 μg cubation for another 30 min at room temperature. Oligomerized of intact mitochondria in 50 μL MSB buffer [400 mM mannitol, Bax was detected by Western blotting. All solutions contained a 50 mM Tris·HCl (pH 7.2), 4 mM MgCl2,10mMKH2PO4,50μM protease inhibitor mixture (Calbiochem) and phosphatase in- rotenone, 5 mM succinate, 5 mg/mL fatty acid free BSA (Sigma)] hibitor (Roche). for 30 min at 30 °C. Then samples were centrifuged at 14,000 × g for 5 min, and supernatants were analyzed for cytochrome c Submitochondrial Fractionation. Mitochondria were isolated, and release by ELISA. submitochondrial fractionation was performed as previously described (1, 2). Briefly, SF767 cells were grown to 95% con- Transmission Electron Microscopy. Specimens for transmission elec- fluence on six 15-cm plates, washed with ice-cold PBS, and tron microscopy (TEM) were fixed with 4% (vol/vol) para- centrifuged at 500 × g for 10 min at 4 °C; then the supernatant formaldehyde and 0.4% glutaraldehyde in 0.1 M sodium cacodylate was removed. The cell pellet was washed with SHE buffer [250 buffer, rinsed with 0.1 M sodium cacodylate and distilled water, and mM sucrose, 10 mM Hepes, 1 mM EGTA (pH 7.4), with pro- dehydrated in ascending grades [50–100% (vol/vol)] of ethanol. tease inhibitor mixture (Calbiochem)] and was centrifuged at Samples were transitioned from 100% (vol/vol) ethanol to a 1:1 500 × g for 10 min at 4 °C. The pellet was resuspended in SHE mixture of 100% (vol/vol) ethanol and LR White resin and then to buffer, homogenized 40 times with a loose-fitting Dounce ho- 100% (vol/vol) LR White hard-grade acrylic resin. Resin samples mogenizer, and centrifuged at 800 × g for 5 min at 4 °C. The were cured at 50 °C. Sections (70-nm) were collected from a Leica resulting supernatant was transferred to a fresh tube, and the UCT ultramicrotome on 200-mesh nickel grids. Sections were pellet containing the nuclear fraction was discarded. The su- stained with uranyl acetate and lead citrate. pernatant was centrifuged at 10,000 × g for 10 min at 4 °C. The For immunogold staining, cellular sections on nickel grids were supernatant containing the cytosolic fraction was removed, and floated on top of drops of 1% lysine, 0.2% sodium metaperiodate, the mitochondrial pellet was resuspended in SHE buffer. An in Tris-buffered saline (TBS) at room temperature for 30 min. aliquot was taken to determine the protein concentration using Grids were washed six times with TBS, twice with 0.05% Tween- the Bradford method. The mitochondria in SHE buffer then 20 + 1% BSA/TBS, and three times with TBS. Sections were were centrifuged at 16,000 × g for 10 min at 4 °C. The mito- incubated in 1% normal goat serum + 2% BSA + 0.2% cold chondrial pellet was resuspended in swelling buffer (10 mM water fish skin gelatin in TBS for 15 min at room temperature KH2PO4, pH 7.4) at a concentration of 200 μg mitochondrial for preblocking and then were rinsed six times in TBS. Sections protein/mL and was incubated on ice for 20 min with gentle were incubated overnight at 4 °C with anti-V5 antibody (In- mixing every 5 min. Then the same volume of shrinking buffer vitrogen) diluted at 1:25 in 2% BSA/TBS. After warming to [10 mM KH2PO4 (pH 7.4), 32% (wt/vol) sucrose, 30% (wt/vol) room temperature, sections were washed three times with 0.05% glycerol, 10 mM MgCl2] was added to the mitochondria in Tween, 1% BSA in TBS, and six times with 1% BSA in TBS. swelling buffer and centrifuged at 10,000 × g for 10 min at 4 °C. Gold-labeled goat anti-mouse, 10-nm size (Electron Microscopy The supernatant containing the outer mitochondrial membrane Services), was diluted 1:25 in 2% BSA/TBS; sections were in- (OMM) and the mitochondrial intermembrane space (IMS) cubated in the gold-labeled secondary solution for 30 min at fractions was transferred to a fresh tube. The mitoplast pellet room temperature. Grids were washed three times with 0.05% was washed by adding a 1:1 mixture of swelling and shrinking Tween-20, 1% BSA/TBS and six times with 1% BSA/TBS and buffer and was centrifuged at 10,000 × g for 10 min at 4 °C. After

Jensen et al. www.pnas.org/cgi/content/short/1316700111 2of9 washing, the mitoplast pellet was resuspended in swelling buffer lipids were visualized using the Typhoon 9410 variable mode and incubated on ice for 20 min with gentle mixing every 5 min. imager and quantified by ImageQuantTL (GE Healthcare). OMM/IMS- and inner mitochondrial membrane (IMM)-con- taining fractions were centrifuged at 14,000 × g for 60 min at 4 °C In Vivo Xenograft Studies. All animals were used under a protocol in an ultracentrifuge (Beckman Coulter). The pellets represent approved by the Institutional Animal Care and Use Committee of OMMs and IMMs, respectively; the supernatant of the OMM/ Northwestern University. Transduced SF767 cells (5 × 105 cells) IMS fraction contains the IMS proteins. The IMS fraction was were injected intracranially into SCID mice (n = 5orn = 10 for concentrated using centrifugal filters (Millipore). The fractions each transfectant). Briefly, cells were detached from culture were resuspended in lysis buffer and mitochondrial membrane flasks and suspended in HBSS. Each mouse was anesthetized markers were detected by Western blotting. and placed in a stereotaxic frame, and the surgical area was cleaned with alcohol and Betadine (Purdue Products L.P.). An CerS Activity Assays. Cells were homogenized in 20 mM Hepes- incision was made in the scalp, and a 0.7-mm burr hole was cre- KOH (pH 7.2), 25 mM KCl, 250 mM sucrose, and 2 mM MgCl 2 ated in the skull with a microsurgical drill 2 mm lateral right of containing a protease inhibitor mixture (Sigma). Protein content was determined using the BCA reagent (Pierce). Homogenates the sagittal suture and 0.5 mm posterior of bregma. A Ham- × 5 were incubated with 15 mM N-(7-nitro-2-1,3-benzoxadiazol-4-yl) ilton syringe was loaded with 5 10 cells and inserted 3.5 mm (2S,3R)-2-aminooctadecane-1,3-diol [NBD-sphinganine into the brain. The cells were implanted over a period of 5 (Avanti Polar Lipids)], 20 mM defatted BSA (Sigma), and 50 mM min, and the needle was left in place for 1 min before the sy- C16-CoA(forCerS6)orC22-CoA(forCerS2)(AvantiPolar ringe was withdrawn. After surgery, the skin was closed with Lipids) for 10–40 min at 37 °C. Lipids were extracted and sutures. For these studies, the cells were implanted in ∼7-wk-old separated by TLC using chloroform, methanol, and 2-M NH4OH female CB17 SCID mice (Taconic Farms). Mice were killed (40:10:1, vol/vol/vol) as the developing solvent. NBD-labeled upon observation of neurological impairment.

1. Lee H, et al. (2011) Mitochondrial ceramide-rich macrodomains functionalize Bax upon 2. Vaseva AV, Moll UM (2013) Identification of p53 in mitochondria. Methods Mol Biol irradiation. PLoS ONE 6(6):e19783. 962:75–84.

A Bcl2L13 BH4 BH3 BH1 BH2 BHNo MA

Bcl-2 Homology domains B Bax CED-9 Mcl-1 NR-13

Bcl-xL Bcl-2 Bcl-w BHRF-1 Bcl-b Boo Bok Bak LMWr-HL Bcl2L12 Bcl2L14 Bcl2L13 Bfl-1 Debcl Bad

C Esophageal adenocarcinoma Oral Cavity Carcinoma

fc=1.87 fc=2.47

Log2 median-centered ratio -5 -6 p=2.63x10 Log2 median-centered ratio p=3.02x10

Duodenum Esophagus Esophageal Cervix Oral Palate Tonsil Oral adenocarcinoma Uteri Cavity Cavity Carcinoma

Fig. S1. Bcl2L13 domain structure and phylogenic analysis expression in solid cancers. (A) Bcl2L13 domain structure including Bcl-2 homology domains 1–4 (BH1–4), the BHNo and membrane anchor (MA) domains. (B) Phylogenic analysis of Bcl2L13 and other Bcl-2 family members. (C) Bcl2L13 mRNA expression in solid malignancies. fc, fold change.

Jensen et al. www.pnas.org/cgi/content/short/1316700111 3of9 A shL13-1 shL13-4 shL13-3 shL13-2 653-671 759-777 1084-1102 2229-2247 Bcl2L13 mRNA coding region

3’UTR

D 60 shCo shL13-2 shL13-3 p<0.05 shL13-4 B 50 shCo shL13-3 kDa shL13-1 shL13-2 shL13-4 75-- -Bcl2L13- 40 75- -Hsp70

30

C shCo shL13-1 shL13-2 shL13-3 shL13-4 % V positive cells Annexin 20 kDa 0 4 8 16 0 4 8 16 0 4 8 16 0 4 8 16 0 4 8 16 [hrs] STS 25- p<0.05 -LS+N aCasp-3 -LS (D175) 10 25- aCasp-7 -LS (D175) 75- 0 -Hsp70 - + STS

Fig. S2. RNAi mulitplicity studies. (A) Binding sites of shRNAs within the Bcl2L13 mRNA and 3′-UTR. (B) Multiple shRNAs targeting Bcl2L13 reduce endogenous levels of Bcl2L13 as shown by anti-Bcl2L13 Western blot analysis. Heat shock protein 70 (Hsp70) is shown as a loading control. (C and D) Multiple Bcl2L13- targeting shRNAs sensitize glioma cells to STS-induced apoptosis as indicated by increased caspase-3 and -7 cleavage (Hsp70 is shown as a loading control) (C) and annexin V positivity (D). LS, large subunit, LS+N, large subunit plus N peptide.

AB CCRF-CEM (ALL) K562 (CML)

CSII-vector CSII-Bcl2L13 shCo shBcl2L13

kDa 0 4 8 16 0 4 8 16 [hrs] STS kDa 0 4 8 16 0 4 8 16 [hrs] STS 20- -LS+N aCasp-3 20- -LS+N aCasp-3 CSII-Vector CSII-Bcl2L13 -LS (D175) shCo shBcl2L13 -LS (D175) - Bcl2L13 - Bcl2L13 20- aCasp-7 - Hsp70 aCasp-7 20- -LS (D175) - Hsp70 -LS (D175)

37- aCasp-9 -p35 aCasp-9 -p35 37- (D232) (D232) 75- 75- -Hsp70 -Hsp70

C CCRF-CEM D 2 CSII-Vector CSII-Bcl2L13 CSII-Vector 0 4 8 16 0 4 8 16 [hrs] STS kDa 1.5 CSII-Bcl2L13 75- 1 -BaxOligo Linear (CSII- 50- 0.5 Vector) Linear (CSII- 37- dimer 0 -Bax Ratio Bax dimer/monomer time Bcl2L13) 25-

Oligomerization 20- monomer 2 -Bax CSII-Vector 20- -COXIV 1.5 15- CSII-Bcl2L13 1 20- -Bax Linear (CSII- 20- 0.5 Vector) Mito

-COXIV Rel. Bax insterion 15- insertion 0 Linear (CSII- time Bcl2L13)

Fig. S3. Bcl2L13 blocks apoptosis in leukemia cells upstream of Bax activation. (A) Effect of enforced Bcl2L13 expression on STS-induced apoptosis in the acute lymphoblastic leukemia (ALL) cell line, CCRF-CEM. (Left) Anti-Bcl2L13 Western blot assessing levels of Bcl2L13 overexpression. Hsp70 is shown as a loading control. (Right) Western blot analysis for active caspases upon STS treatment. Hsp70 is shown as a loading control. (B) Impact of shRNA-mediated knockdown of Bcl2L13 on STS-induced apoptosis in the chronic myelogenous leukemia (CML) cell line, K562. (Left) Anti-Bcl2L13 Western blot confirms reduction in en- dogenous Bcl2L13 protein levels. (Right) Western blot analysis for active caspases upon STS treatment. (C) Bcl2L13 inhibits apoptosis progression upstream of Bax activation in CCRF-CEM cells ectopically expressing Bcl2L13. (Upper) Bax dimer- and oligomerization assay. (Lower) The ability of Bax to insert into the mitochondrial membrane in response to STS was assessed by anti-Bax Western blot analysis. Migration positions of monomeric, dimeric, and oligomeric Bax are indicated. COXIV is shown as a mitochondrial marker and loading control. (D) Densitometric quantification of Bax dimerization and membrane insertion. The intensities of bands representing Bax dimers, monomers, and membrane-inserted monomers were normalized to COXIV. LS, large subunit, LS+N, large subunit plus N peptide.

Jensen et al. www.pnas.org/cgi/content/short/1316700111 4of9 A B HA-Vector HA-CerS2 HA-CerS6

kDa 0 4 8 16 0 4 8 16 0 4 8 16 [hrs] STS 20- -LS+N aCasp-3 kDa HA-Vector HA-CerS2 -LS (D175) 37- -CerS2HA 75- -Hsp70 aCasp-7 20- -LS (D175)

37- -p35 aCasp-9 (D232) 75- -Hsp70 kDa HA-Vector HA-CerS6 37- -CerS6HA 75- -Hsp70

C D siCo siCerS2 siCerS6 kDa 0 4 8 16 0 4 8 16 0 4 8 16 [hrs] STS 20- aCasp-3 -LS+N (D175) -LS

kDa siCo siCerS2 siCerS6 20- -LS aCasp-7 37- -CerS2 (D175) 37- 37- -CerS6 -p35 aCasp-9 (D232) 75- 75- -Hsp70 -Hsp70

E F 150

p<0.05 100

kDa shCo shCerS6 shCo -CerS6 37- 50

75- Percent survival -Hsp70 shCerS6

0 0 204060 Days

Fig. S4. CerS2 and CerS6 are proapoptotic proteins. (A and B) Effect of transient overexpression of CerS2HA and CerS6HA on STS-induced apoptosis in SF767 cells as measured by caspase-3, -7, and -9 cleavage. (A) Anti-HA Western blot analysis confirms overexpression of CerS enzymes. Hsp70 is shown as a loading control. (B) Western blot analysis surveying postmitochondrial caspase activation. (C and D) Effect of siRNA-mediated knockdown of CerS2 and CerS6 on STS- induced apoptosis in SF767 cells as measured by caspase-3, -7, and -9 cleavage. (C) Western blot analysis for endogenous CerS proteins shows robust KD of CerS2 and CerS6, respectively. (D) Caspase-9, -3, and -7 activation was monitored by Western blot analysis. Hsp70 is shown as a loading control. LS, large subunit, LS+N, large subunit plus N peptide. (E) Western Blot analysis of SF767 cells stably expressing CerS6-targeting shRNA. Hsp70 is shown as a loading control. (F) Kaplan–Meier survival curves of mice xenografted with shCo- or shCerS6-expressing SF767 cells.

Jensen et al. www.pnas.org/cgi/content/short/1316700111 5of9 A Merge/DAPI HA-CerS2 Bcl2L13V5

10 m Zoom

B OMM IMS IMM 75- -Bcl2L13

37- -CerS2

37- -CerS6

15- -cyto c (IMS)

10- -COX17 (IMS) OMM, outer mitochondrial membrane -COXIV (IMM) 15- IMS, intermitochondrial membrane space 25- -Bcl-2 (OMM) IMM, inner mitochondrial membrane

Fig. S5. Bcl2L13 colocalizes and cocompartmentalizes with CerS2 and CerS6. (A) Confocal immunofluorescence microscopy of SF767 cells cotransfected with CerS2HA (green) and V5-tagged Bcl2L13 (red). Overlay is indicated by yellow color. DAPI is shown as a nuclear counterstain. (B) Western blot analysis of submitochondrial fractions for Bcl2L13, CerS2, and CerS6. Bcl-2, cytochrome c, COX17, and COXIV are shown as markers for OMMs, IMS, and IMMs, respectively.

Jensen et al. www.pnas.org/cgi/content/short/1316700111 6of9 A WT BH4 BH3 BH1 BH2 BHNo MA

BHNo BH4 BH3 BH1 BH2 MA

B WCL C Flag-Bcl2L13 Flag- BHNo D Flag-Bcl2L13 Flag- BHNo FLAG FLAG BHNo IgG IgG IgG input input IgG input input anti-FLAG anti-FLAG

- * anti-FLAG anti-FLAG

kDa Bcl2L13 kDa kDa 75- - - IgG -WT - IgGH 50- H 50- HA - CerS2HA -CerS6 - * 37- 37- 50- - * 25- 25- - IgGL - IgGL 37- - * Co-expression: HA-CerS2+ FLAG-Bcl2L13 wt/mt Co-expression: HA-CerS6+ FLAG-Bcl2L13 wt/mt IP: anti-FLAG / WB: anti-HA IP: anti-Flag / WB: anti-HA 25- - BHNo V5 E V5 F BHNo Vector kDa Bcl2L13 75- -WT VectorV5 Bcl2L13V5 BHNoV5 kDa 0 4 8 16 0 4 8 16 0 4 8 16 hrs [STS] 50- 20- -LS+N aCasp-3 - * 15- -LS (D175) 37- -LS aCasp-7 WB: anti-V5 - * - BHNo 20- (D175) 25- - * 20- 75- -Hsp70 75- -Hsp70

G VectorV5 Bcl2L13V5 BHNoV5 kDa 0 0.5 1 2 4 0 0.5 1 2 4 0 0.5 1 2 4 [hrs] curcumin 100-

HA 75- -CerS6 dimer

50- HA 37- -CerS6

3 3 3

2.5 2.5 2.5

2 2 2

1.5 1.5 1.5 Ratio monomer/dimer relative to untreated cells 1 1 1

Fig. S6. Bcl2L13-CerS interaction is mediated by the BHNo domain. (A) Schemes of wild-type Bcl2L13 and a deletion mutant lacking the BHNo domain (ΔBHNo). Dotted lines indicate the deleted region. (B–D) Coimmunoprecipitation of Bcl2L13Flag or ΔBHNoFlag and CerS2HA or CerS6HA. IP: Flag; immunoblot: anti-HA. WCL, whole-cell lysate. (E) Western blot analysis to assess expression levels of Bcl2L13V5 (∼75 kDa) and ΔBHNoV5 (∼27 kDa). Asterisks indicate non- specific background bands. (F) Effect of Bcl2L13V5 and ΔBHNoV5 transient overexpression on STS-induced apoptosis compared with V5-vector alone. Caspase-3 and -7 activation was monitored by Western blot analysis. Hsp70 is shown as a loading control. LS, large subunit, LS+N, large subunit plus N peptide. (G)(Upper) Western blot showing the effect of curcumin-induced CerS6HA dimerization in cells cotransfected with V5-vector alone (Left), Bcl2L13V5 (Center), or ΔBHNoV5 (Right). (Lower) Densitometric analysis of CerS6HA dimer formation corresponding to the Western blots in the upper panel.

Jensen et al. www.pnas.org/cgi/content/short/1316700111 7of9 A

kDa Input anti-Bcl-2 IgG

37- - Cer2

37- -CerS6

25- - Bcl-2 IP: anti-Bcl-2 WB: anti-CerS2 or CerS6 B L IgG Input kDa anti-Bcl-x

37- - Cer2

37- - Cer6

25- - Bak

25- - Bcl-XL

IP: anti-Bcl-xL WB: anti-Bak WB: anti-CerS2 or CerS6

Fig. S7. Canonical members of the Bcl-2 protein family lacking a BHNo domain do not interact with CerS2 or CerS6. (A and B) Anti-CerS2 and CerS6 Western blot analysis of anti Bcl-2 (A) and anti–Bcl-xL immunoprecipitates (B). A Western blot for Bak, a known Bcl-xL interacting protein, is shown as a positive control.

Apoptosis progression

CerS homo- and heterodimerization Ceramide Bax oligomerization A. Normal Tissue production and Cyto c release Apoptosis stimulus Cer Cer cytosol

Bax Bax CerS2 CerS6 CerS6 CerS6 CerS2 CerS2 CerS6 OMM CerS2

Cyto c IMS

matrix IMM

B. Bcl2L13 Overexpressing Tumors cytosol

Bax Bax Bcl2L13CerS6 CerS2 Bcl2L13 OMM

Cyto c IMS

matrix IMM

Fig. S8. Scheme of the proposed modus operandi of Bcl2L13. (A) In normal tissue, when an apoptotic stimulus is sensed, CerS2 and CerS6 homo- or heter- odimerize to produce ceramide, which is necessary for Bax oligomerization and cytochrome c release. (B) In tumors with overexpression of Bcl2L13, Bcl2L13 binds to CerS2 and CerS6, preventing the homo- and heterodimerization required of the CerSs to produce ceramide, leading to no oligomerization of Bax and no cytochrome c release necessary for apoptosis. cyto c, cytochrome c.

Jensen et al. www.pnas.org/cgi/content/short/1316700111 8of9 Table S1. Bcl2L13-interacting proteins as revealed by yeast two-hybrid screen Gene Full name Accession no.

PPT2 Palmitoyl-protein thioesterase 2 NM_138717.2 NR2C2AP Nuclear receptor 2C2-associated protein NM_176880.4 BAZ2B Bromodomain adjacent to zinc finger domain NM_013450.2 PCBP1 Poly(rC) binding protein 1 NM_006196.3 ARL6IP1 ADP ribosylation factor-like 6 interacting protein 1 NM_015161.1 NRBP1 Nuclear receptor binding protein 1 NM_0133392.2 LAYN Layilin NM_001258390.1 HNRNPK Heterogeneous nuclear ribonucleoprotein K NM_002131.2 JPH4 Junctophilin 4 NM_177049.5 NDRG4 NDRG family member 4 NM_001130487.1 MGMT O-6-methylguanine-DNA methyltransferase NM_002412.3 NETO2 Neuropilin (NRP) and tolloid (TLL)-like 2 NM_018092.4 ACP5 Acid phosphatase 5, tartrate resistant NM_001111035.1 LASS2 (CerS2) LAG1 homolog, ceramide synthase 2 NM_181746.3 SH3RF3 SH3 domain containing ring finger 3 NM_001099289.1 SRSF3 Serine/arginine-rich splicing factor 3 NM_003017.4 RPN2 Ribophorin II NM_002951.3 IDI1 Isopentenyl-diphosphate delta isomerase 1 NM_004508.2 SQLE Squalene epoxidase NM_003129.3 BCL9 B-cell CLL/lymphoma 9 NM_004326.3 FUS Fused in sarcoma NM_004960.3 ETNK2 Ethanolamine kinase 2 NM018208.2 (p35) (CDK5R1) Cyclin-dependent kinase 5, regulatory subunit 1 NM_003885.2 GTF2F1 General transcription factor IIF, polypeptide 1 NM_002096.2 SMARCD3 SWI/SNF related, matrix associated, actin dependent NM_001003802.1 regulator of chromatin, subfamily d, member 3 TUBB β-Tubulin NM_178014.2 UBC Ubiquitin C NM_021009.5 CAMLG Calcium-modulating ligand NM_001745.3 CDC42 Cell division cycle 42 NM_044472.2

Jensen et al. www.pnas.org/cgi/content/short/1316700111 9of9