71-8700

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of MDA-MB-231 (Lane 1), NTERA-2 (Lane 2), MCF-7 (Lane 3), A549 (Lane 4), and Caco-2 (Lane 5). The blots were probed with Anti- CUL1 Rabbit Polyclonal Antibody (Product # 71-8700, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A ~ 89 kDa band corresponding to CUL1 was observed across cell lines tested. Additionally ~68 kDa band was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with Pierce™ Power Blotter System (Product # 22834). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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Experimental details: Western blot analysis of Cullin-1 was performed by loading 10 µg of whole cell lysates from DMSO, MG132 or MLN4924 treated U2OS cells onto a SDS PAGE gel. Proteins were transferred, blocked and endogenous Cullin-1 was detected using a CUL-1 polyclonal antibody (Product # 71-8700) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of MDA-MB-231 (Lane 1), NTERA-2 (Lane 2), MCF-7 (Lane 3), A549 (Lane 4), and Caco-2 (Lane 5). The blots were probed with Anti- CUL1 Rabbit Polyclonal Antibody (Product # 71-8700, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A ~ 89 kDa band corresponding to CUL1 was observed across cell lines tested. Additionally ~68 kDa band was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with Pierce™ Power Blotter System (Product # 22834). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 ATP-depletion resulted in the inhibition of CSN deneddylase activity. a 10 nM His-6-Cul1/ROC1 were pre-neddylated for 30 min at 30degC. Then 10 mug HeLa cells extract were added at 30degC (time plot: lanes 3 - 6 ); or alternatively, ATP-trap mix was added for 45 min at 30degC, followed by the addition of 10 mug HeLa cells extract at 30degC (time plot: lanes 8 - 11 ). b 3 nM His-6-Cul1/ROC1 was pre-neddylated for 30 min at 30degC. Then 100 nM purified CSN was added at 20degC (time plot: lanes 3 - 6 ); or alternatively, ATP-trap mix was added for 45 min at 30degC, followed by the addition of 100 nM purified CSN at 20degC (time plot: lanes 8 - 11 ). c 10 nM His-6-Cul1/ROC1 was incubated as described in b ; time plots were for 1.5-7.5 min, and not for 5-40 min as in b . Blotting was directed against NEDD8, and not against Cul1, as described elsewhere.

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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Endogenic Cul1 deneddylation is enhanced during simultaneous neddylation. a 20 mug HeLa cells extract were incubated for 30 min at 30degC ( lanes 2 - 7 ) or with the addition of ATP-trap mix prior to the incubation ( lanes 8 - 13 ). Simultaneous ATP-depletion was performed in lanes 3 - 7 after 30 min incubation, on a time plot of 1.5-7.5 min at 30degC. Simultaneous neddylation was performed in lanes 9 - 13 after 30 min, by the addition of AMP-PNP, on a time plot of 1.5-7.5 min at 30degC. b 10 nM His-6-Cul1/ROC1 was neddylated at 30degC on a time plot of 2.5-30 min, without or with the addition of ATP-trap mix ( lanes 2 - 6 and lanes 7 - 11 , respectively). In lane 12 , the neddylation mix was added to the reaction after pre-incubation with ATP-trap mix. c 10 nM His-6-Cul1/ROC1 was neddylated at 30degC using AMP-PNP on a time plot of 2.5-30 min, either directly ( lanes 2 - 6 ) or after pre-incubation with ATP-trap mix ( lanes 7 - 11 ).

Supportive validation

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Experimental details: Figure 2 Effect of MIB on p27, SKP2, and other cell cycle regulators ( A ) LNCaP cells were incubated with 10 ng/ml MIB for the indicated periods. Cell lysate was harvested and expression of the indicated proteins was assessed by immunoblotting with respective antibodies. Tubulin is shown as a loading control. SKP2 and p27 protein levels were quantitated using a demonstration copy of the Totallab software from Nonlinear Dynamics. ( B ) LNCaP cells were incubated with the indicated concentrations of MIB for 72 h, followed by preparation of total cell lysate and immunoblotting with antibodies against p27, SKP2, and tubulin. The effects of androgen on p27 and SKP2 were maximal at 1 ng/ml MIB. ( C ) LNCaP cells were maintained in the presence of 1 ng/ml MIB for 96 h after which the medium was replaced and 750 ng/ml of the antiandrogen cyproterone acetate (CA) was added. Cell lysates were prepared after the indicated times, and p27 and SKP2 levels were assessed by immunoblotting. ( D ) LNCaP cells were incubated with 1 ng/ml MIB and/or 750 ng/ml CA for 72 h and cell lysates were prepared. Expression of the indicated proteins was assessed by immunoblotting. Tubulin levels are shown as loading controls. ( E ) LNCaP cells were incubated with 1 ng/ml MIB and/or 750 ng/ml CA for 72 h and cells were fixed for flow cytometry. Cell cycle profiles and the fraction of cells in each cell cycle phase are shown. ( F ) LNCaP cells were maintained in the absence or presence of 10 ng/ml MIB for 7

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Experimental details: Figure 3 CSN6 enhances Fbxw7's autoubiquitination and degradation (a) CSN6 increased Fbxw7alpha turnover. 293T cells were infected with lentivirus to express CSN6 shRNA or luciferase control shRNA. The cells were then transfected with Flag-Fbxw7alpha and were then treated with cycloheximide (CHX; 100 ug/ml) for the indicated times. The density of Fbxw7alpha was measured and the integrated optical density (OD) was measured. The turnover of Fbxw7alpha is indicated graphically. (b) CSN6-mediated Fbxw7alpha downregulation was proteasome-dependent. 293T cells were cotransfected with indicated plasmids and were treated with or without 50 ug/ml MG132 for 6 hours. The cell lysates were then immunoblotted with the indicated antibodies. (c) CSN6 enhanced Fbxw7alpha ubiquitination through lysine 48 linkage. 293T cells were cotransfected with Flag-Fbxw7alpha with or without GFP-CSN6 plus His-ubiquitin wild type (wt), K63R mutant, or K48R mutant. Cells were treated with 50 ug/ml MG132 for 6 hours before harvest. The ubiquitinated Fbxw7alpha proteins were pulled down using Ni-NTA-agarose beads and detected with anti-Flag antibody. (d) An F-box domain deletion mutant of Fbxw7alpha was resistant to CSN6-mediated ubiquitination. 293T cells were cotransfected with GFP-CSN6, His-ubiquitin plus Flag-Fbxw7alpha wild-type or F-box domain deletion mutant (DeltaF). Cells were treated with 50 ug/ml MG132 for 6 hours before harvest. The ubiquitinated Fbxw7alpha proteins were pulled down using Ni-

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 ATP-depletion resulted in the inhibition of CSN deneddylase activity. a 10 nM His-6-Cul1/ROC1 were pre-neddylated for 30 min at 30degC. Then 10 mug HeLa cells extract were added at 30degC (time plot: lanes 3 - 6 ); or alternatively, ATP-trap mix was added for 45 min at 30degC, followed by the addition of 10 mug HeLa cells extract at 30degC (time plot: lanes 8 - 11 ). b 3 nM His-6-Cul1/ROC1 was pre-neddylated for 30 min at 30degC. Then 100 nM purified CSN was added at 20degC (time plot: lanes 3 - 6 ); or alternatively, ATP-trap mix was added for 45 min at 30degC, followed by the addition of 100 nM purified CSN at 20degC (time plot: lanes 8 - 11 ). c 10 nM His-6-Cul1/ROC1 was incubated as described in b ; time plots were for 1.5-7.5 min, and not for 5-40 min as in b . Blotting was directed against NEDD8, and not against Cul1, as described elsewhere.

Supportive validation

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Experimental details: Figure 2 CSN6 regulates SCF (Skp1/Cullin-1/Fbxw7) complex to stabilize Myc (a) CSN6 associated with Myc, Fbxw7 and Cullin-1. The U2OS cell lysates were immunoprecipitated with anti-CSN6 and immunoblotted with indicated antibodies. (b) CSN6 knockdown led to decreased Cullin-1 neddylation. The U2OS cell lysates were immunoprecipitated with anti-Cullin-1, followed by immunoblotting with indicated antibodies. (c) CSN6 expression was inversely related to Fbxw7 expression. Immunoblot analysis of Myc and Fbxw7alpha in CSN6-overexpressing or CSN6-knockdown cells. Cell lysates were analyzed by indicated antibodies. (d) Knockdown of CSN6 increased the expression of Fbxw7 and reduced Myc. Indicated cells were infected with lentivirus to express one of two independent CSN6 shRNAs (#1, #2) or luciferase control shRNA. Cell lysates were immunoblotted with indicated antibodies. (e) CSN6 reduced steady-state expression of Fbxw7 to upregulate Myc. Indicated cells were transfected with increasing amounts of GFP-CSN6. Cell lysates were immunoblotted with indicated antibodies. Fbxw7alpha and Fbxw7beta isoforms were identified. (h) Knockdown of CSN6 enhanced Myc ubiquitination. Indicated cells were infected with lentivirus to express one of two independent CSN6 shRNAs (#1, #2) or luciferase control shRNA. The cells were then transfected with His-ubiquitin and treated with MG132 for 6 hours before harvest. The ubiquitinated Myc proteins were pulled down using Ni-NTA-agarose beads and detected with

Supportive validation

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Experimental details: Figure 4 CSN6 enhances neddylation of Cullin-1 via its MPN domain (a) Neddylation status of Cullin in gel filtration chromatography fractions from spleen extracts from Csn6 +/+ (wt) and Csn6 +/- mice. Extracts of spleen from CSN6 +/+ or Csn6 +/- mice (4 weeks old) were ground and subjected to lysis. Lysates were fractionated by gel filtration chromatography. Fractions were resolved by SDS-PAGE, followed by immunoblotting with indicated antibodies. Molecular size of eluted fraction is indicated above. (b) Non-neddylated Cullin was increased in Csn6 +/- mice. Extracts of spleen from CSN6 +/+ (wt) or Csn6 +/- mice were fractionated by gel filtration chromatography. Representative fractions 36-54 from (a) were resolved by SDS-PAGE, followed by immunoblotting with anti-Cullin-1, anti-CSN6, and anti-CSN5 antibodies. (c) CSN6 competed with CSN5 for binding to Cullin-1 and affected Cullin-1 neddylation. 293T cells were transfected with His-Cullin-1 plus increasing amounts of GFP-CSN6 or Myc-CSN5. The neddylated Cullin-1 proteins were pulled down using Ni-NTA-agarose beads and detected with anti-Nedd8 antibody. The lysates were also immunoprecipitated with CSN6 antibody or CSN5 antibody and were subjected to immunoblotting with anti-Cullin-1. (d) MPN domain of CSN6 had an important role in competing with CSN5 for Cullin-1 binding and affected Cullin-1 neddylation. 293T cells were cotransfected with indicated plasmids. The lysates were immunoprecipitated with anti-Flag or anti-CSN5 ant

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Experimental details: Figure 6 CSN6 haplo-insufficiency delays the onset/progression of Myc-mediated lymphomagenesis (a) Survival of Eu- Myc mice was prolonged by CSN6 haplo-insufficiency. The genotypes of the transgenic mice are indicated next to the Kaplan-Meier survival curves. The numbers of mice analyzed in each group are denoted. (b) CSN6 haplo-insufficiency caused low levels of Cullin neddylation and low expression of Myc. Lymphoma tissues from Eu- Myc / Csn6 +/+ (1-10) and Eu- Myc / Csn6 +/- (11-25) mice were immunoblotted with the indicated antibodies. (c) Levels of Fbxw7 were elevated in lymphomas from Eu- Myc / Csn6 +/- mice . Lymphomas arising from mice from (b) underwent IHC staining. Representative photographs of Fbxw7 and H&E staining are shown. (d) Levels of neddylated Cullin and Myc were reduced and level of Fbxw7 was increased in lymphomas from transgenic mice. Levels of indicated proteins in lymphomas arising from mice in (b) and (c) were quantitated by integrating optical density from Image J and are demonstrated as bar graphs. Each result shown is representative of 3 independent experiments. Error bars represent 95% CI; Student t-test ,* P< 0.001. (e) The model depicts the pivotal role of the CSN6-Cullin-Fbxw7 axis in Myc-induced lymphomagenesis.

Supportive validation

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Experimental details: Figure 2 CSN5 inhibition results in the inactivation of a subset of CRLs and the stabilization of their substrates. Effect on CRL components and substrates after treatment of HCT116 cells with either 1 muM MLN4924 or 1 muM CSN5i-3. ( a ) Immunoblottings for Cul1 and its CRL components Rbx and Skp1; ( b ) for cullins Cul2, Cul3 and Cul4A; ( c ) for F-box protein Skp2 and the SCF Skp2 substrates p21 and p27; ( d ) for F-box proteins Fbxo22 and Fbxo30; ( e ) for F-box protein betaTrCP and the SCF betaTrCP substrates Wee1 and pIkappaBalpha; ( f ) for the adaptor KEAP1 and the Cul3 KEAP1 substrate Nrf2; ( g ) for F-box proteins Fbxw7 and Fbxo3.

Supportive validation

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Experimental details: FIGURE 4. A candidate beta -TRCP phosphodegron in SPAR. A , schematic representation of SPAR domains and SPAR fragments: actin-binding domains ( Act1 and Act2 ), RapGAP, PDZ, and guanylate kinase binding ( GKBD ) domain. The candidate DSGIDT phosphodegron motif (residues 1304-1309) identified in the Act2 domain of SPAR closely resembles the consensus beta-TRCP phosphodegron ( inset ). Boundaries of generated C-terminal fragments of SPAR are depicted; C-1, C-2, and Act2 fragments contain the putative phosphodegron motif, whereas C-3 does not. B and C , SPAR fragments spanning the beta-TRCP phosphodegron bind to beta-TRCP. HEK293T cells were transfected with vectors expressing SPAR fragments C-1, C-2, C-3 ( B ) or Act2 ( C ) (0.6 mug) either alone or with pCMV-GST or pCMV-GST-beta-TRCP (0.6 mug). In addition, pCMV-HA-Plk2 WT/K108M (0.6 mug) and DN Cul1 (2 mug) were co-transfected as indicated. After 24 h, cell extracts were used for GSH-Sepharose pulldown assays, and proteins were immunoblotted with Myc antibodies. Crude lysates were blotted as an input control. D and E , phosphodegron-dependent binding to beta-TRCP. Constructs expressing point mutations (S1305A, T1309A) in full-length SPAR ( E ) and the Act2 fragment ( D ) (myc-SPAR AA or myc-Act2 AA , 0.6 mug) were transfected into HEK293T cells along with pCMV-HA-Plk2 WT/D201A (0.6 mug), pCMV- DN Cul1 (2 mug), and pCMV-GST or pCMV-GST-beta-TRCP (0.6 mug) as indicated. Cell lysates were incubated with GSH-Sepharose and immuno

Supportive validation

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Experimental details: Figure 1 CSN5i-3 is a potent inhibitor of CSN5-catalysed cullin deneddylation. ( a ) Schematic illustration of the CRL cycle and the role of cullin neddylation and deneddylation. Ad, adaptor protein; SRM, interchangeable substrate recognition module; Sub, substrate. ( b ) Chemical structures of CSN5 inhibitors illustrating the optimization of the high throughput screening (HTS) hit CSN5i-1a to the cell active intermediate CSN5i-2, its R,R -enantiomer CSN5i-2e and to the final compound CSN5i-3. ( c ) Co-crystal structures of CSN5 with CSN5i-1b and CSN5i-3. The structures revealed the direction for compound extension into the substrate binding cleft of CSN5 (red arrow). ( d ) Dose-response curve for inhibition of CSN activity by CSN5i-3 as measured in a biochemical enzyme assay ( n =2, +-s.d.). ( e ) Immunoblotting for Cul1 after treatment of HCT116 cells with 1 muM CSN5i-3 and for Cul1 and CSN5 after treatment with CSN5 siRNA.

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Experimental details: Figure 4 CSN5i-3 inhibits tumour growth of a human xenograft. ( a ) SU-DHL-1 xenografts were grown in SCID-bg mice and dosed by oral administration with either vehicle control or CSN5i-3 at the indicated doses and schedules. Mean tumour volumes are shown+-s.e.m. ( n =4; P

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Experimental details: 10.1371/journal.ppat.1009936.g001 Fig 1 Entamoeba histolytica promotes the degradation of cullin-1/5 from THP-1 derived macrophages, colonic epithelial cells, and proximal colonic loop tissues in a time- and dose-dependent fashion. (A) Schematic representation of Skp-1-Cullin-1-F-box (SCF) protein E3 complex (Skp-1: S-phase kinase-associated protein 1; Rbx: RING [Really Interesting New Gene]-box1; E2: Ubiquitin-loaded E2). (B, C) THP-1 macrophages were incubated with different Eh :macrophage ratios, or (D, E) for increasing times (5 to 30 min) with Eh (20:1 ratio) and Eh (10:1 ratio). (F, G) T84 colonic epithelial cells were grown in 12-well plates and stimulated with Eh (10:1 ratio) for 5 to 30 min. (H, I) Proximal colonic loops were inoculated with Eh and cell lysates were prepared. Post Eh stimulations, cells were washed and cytoplasmic extracts were prepared, and an equal amount of protein was loaded on to the SDS- PAGE gel (7.5%) and immunoblotted with the anti-cullin-1, anti-cullin-5 and anti-GAPDH antibody. Highlighted boxed areas on the figures show point of interest for cullin-1/5 as described in text. (J) Protein-protein interaction using STRING v11 showed direct interaction between cullin-1, cullin-5, cullin-4A, cullin-4B and Nedd8. Data (B, C, D, E, H, and I) are representative of three independent experiments and data (F and G) are representative of two independent experiment and statistical significance was carried out with Students t- test. Bar represent mean +

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Experimental details: 10.1371/journal.ppat.1009936.g005 Fig 5 Caspase-1 activation during Eh- macrophage contact degrades cullin-1/5. ( A, B) THP-1 derived macrophages were pre-incubated with the pan-caspase inhibitor Z-VAD-fmk (100muM) and caspase-1 specific inhibitor Z-YVAD-fmk (100muM) for 1 h followed by stimulation with Eh (10:1 ratio) for 10 min. Cleavage of the cullin-1/5 were assessed by western blot. (C, D) Wild type (WT) THP-1 and CASP1 CRISPR/Cas9-KO macrophages were stimulated with Eh (10:1 ratio) for 10 min. (E, F) WT, THP-1 def ASC and NLRP3 CRISPR/Cas9-KO THP-1 macrophages were stimulated with Eh (10:1 ratio) for 10 min. After incubation, cells were washed and lysed. Equal amount of protein was loaded on to the SDS-PAGE (7.5%) and immunoblotted with anti-cullin-1 ( Panel: A, C, and E ) , anti-cullin-5 (Panel: B, D and F) and anti-GAPDH antibody. (G) Cullin-1 was immunoprecipitated (Cul-1 IP) using anti cullin-1 antibody and post immunoprecipitation it was incubated with recombinant caspase-1 (C-1) for 4h or overnight (O/N). (H) Immunoprecipitated cullin-1 was incubated with recombinant caspase-1 or with Y-VAD-fmk (50muM or 100muM) or Z-VAD-fmk (100muM). (I) Immunoprecipitated cullin-5 was incubated with recombinant caspase-1 or with Z-VAD-fmk (100muM) or Y-VAD-fmk (100muM). (J) Recombinant cullin-1 (rec Cul-1) was incubated with recombinant caspase-1 overnight at 37deg and was immunoblotted with anti-cullin-1 antibody. Note, recombinant cullin-1 (rec Cul-1) has a molecular weight of

Supportive validation

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Experimental details: Figure 1 Loss of CSN6 leads to destabilization of Myc and decreases Myc transcriptional activity (a) Immunoblot analysis of Csn6 +/- MEFs. Primary MEF cells were prepared from 13.5-day embryos derived from wild-type (wt) Csn6 +/+ and Csn6 +/- mice. After 4 days of culture, cell lysates were analyzed by indicated antibodies. (b) Quantitative RT-PCR analysis of mRNA levels for indicated c-Myc target genes in CSN6-transfected U2OS cells or Csn6 +/- MEFs. The quantitated mRNA expression level was normalized to GAPDH mRNA. Two MEFs were examined for each genotype. Expression level of transfected CSN6 is indicated as numbers on the heat map, and the heat map depicts the natural logarithm of fold-change in mRNA expression. (c) CSN6 knockdown abrogated serum-induced elevation of Myc. Csn6 +/+ MEFs and Csn6 +/- clone 1 (C1) and clone 2 (C2) MEFs were serum-starved for 24 hours and then were refed with serum for 24 hours. Lysates were analyzed by indicated antibodies. (d) CSN6 decreased Myc turnover. 293T cells were transfected with Flag-CSN6 and were then treated with cycloheximide (CHX; 100 ug/ml) for the indicated times. The immunoblot of Myc signal at each time point was measured using a densitometer and the integrated optical density of Myc was measured. The turnover of Myc is indicated graphically. (e) CSN6 expression affected Myc turnover. 35 S-pulse-labeled HA-Myc protein was immunoprecipitated from indicated transfected 293T cell lysates. The mixture was separated by sodium do

Supportive validation

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Experimental details: Fig. 4 Endogenic Cul1 deneddylation is enhanced during simultaneous neddylation. a 20 mug HeLa cells extract were incubated for 30 min at 30degC ( lanes 2 - 7 ) or with the addition of ATP-trap mix prior to the incubation ( lanes 8 - 13 ). Simultaneous ATP-depletion was performed in lanes 3 - 7 after 30 min incubation, on a time plot of 1.5-7.5 min at 30degC. Simultaneous neddylation was performed in lanes 9 - 13 after 30 min, by the addition of AMP-PNP, on a time plot of 1.5-7.5 min at 30degC. b 10 nM His-6-Cul1/ROC1 was neddylated at 30degC on a time plot of 2.5-30 min, without or with the addition of ATP-trap mix ( lanes 2 - 6 and lanes 7 - 11 , respectively). In lane 12 , the neddylation mix was added to the reaction after pre-incubation with ATP-trap mix. c 10 nM His-6-Cul1/ROC1 was neddylated at 30degC using AMP-PNP on a time plot of 2.5-30 min, either directly ( lanes 2 - 6 ) or after pre-incubation with ATP-trap mix ( lanes 7 - 11 ).