Plasmids, protein, antibodies
Human Cul3 and Klhl12 were cloned into pcDNA4 and pcDNA5 vectors for expression in mammalian cells. Cul3, Sec31A and Sec13 were also cloned into pCS2 vector for IVT/T and expression in mammalian cells. pcDNA4-Cul3N250 contains the first cullin repeat of the N-terminal Cul3 (1-250aa) which is sufficient for binding BTB proteins, but not Rbx1 and serves as a dominant negative for Cul3/BTB-mediated ubiquitination. The Klhl12 mutants FG289AA, RL342AA, RGL369AAA, RE416AA, YDG434AAA and RCY510AAA were made by site-directed mutagenesis.
Cul3 and Rbx1 were cloned into pFastBac, co-expressed in Sf9 ES insect cells using the Bac-to-Bac baculovirus expression system (Invitrogen) and purified as a complex by Ni-NTA agarose (Qiagen). Similarly, the Sec31A/Sec13 heterodimer and UBA1 were purified from Sf9 ES insect cells. UbcH5c and Ubc12 were cloned into pQE vector and purified from BL21(DE3) bacterial cells. Ubiquitin was cloned into pET and pCS2 vector with a N-terminal 6xHis tag. The pET-His-ubiquitin was used for bacterial purification whereas pCS2-His-ubiquitin was expressed in mammalian cells. Wildtype ubiquitin, APPBP1-UBA3 and NEDD8 were purchased from Boston Biochem.
To purify recombinant Klhl12 for ubiquitination assays, we expressed pMAL-TEV-Klhl12-his and pMAL-TEV-Klhl12FG289AA-his in BL21(DE3) cells, purified the proteins on amylose resin, cleaved them by TEV protease, and re-purified them on Ni-NTA agarose. WT-Klhl12 and mutants were also cloned into pMAL vector and purified as MBP-tagged proteins for in-vitro protein binding assays.
All shRNAs were cloned in pSuper-GFP neo vector (from Oligoengine) into BglII and XhoI sites. The GFP-Bcl2-Cb5 construct, a fusion between Bcl2 and cytochrome b5, was purchased from Clontech.
We raised mouse monoclonal antibodies against human Klhl12 and human Klhl13. Both antibodies are available at Promab Biotechnologies (cat. # 30058 and # 30067). We also raised antibodies against Sec13, Sec24C, and Sec24D. Other antibodies used in this study are: Cul3 (Bethyl Laboratories, cat. # A301-109A), Sec31A (BD Biosciences, cat. # 612350), Collagen IV (Abcam, cat. # ab19808), anti-FLAG (Sigma, cat. #F3165, #F7425), Ubiquitin (Santa Cruz, cat. # sc-8017, P4D1), Rhodomine phalloidin (Invitrogen, cat. # R415), PDI (1D3) (Assay Designs, cat. #SPA-891), anti LC-3 (Sigma, Cat # L-7543), anti-alpha tubulin (DM1A, Abcam, Cat # ab7291), anti-fibronectin (Abcam, ab2413), anti-GM130 (BD Biosciences, cat. # 610822), and anti-EGFR (Ab12, Neomarkers, MS-400P1). LF-67 (Anti sera for Type I procollagen) was obtained as a generous gift from Dr. Larry Fisher.
The D3 mouse embryonic stem cells (mESC) were maintained in ESC medium containing 15% FBS, 1x sodium pyruvate, 1x NEAA, 1mM β-ME and 1000u/ml LIF (Millipore, cat. # ESG1107) in GIBCO Dulbecco’s Modified Eagle Medium, and grown on 0.1% gelatin-coated tissue culture plates. Hela cells, 293T cells, 3T3 cells and IMR90 cells were maintained in DMEM plus 10% FBS. Dialyzed FBS was bought from Hyclone. The doxycycline-inducible 293T Trex Klhl12-3xFLAG stable cell line was made with Flp-In™ T-REx™ 293 Cell Line system from Invitrogen. Stable cell lines expressing other BTB-proteins were generated accordingly. These cell lines were maintained with 10% TET(-) FBS, blasticidin and hydromycin B as instructed and expression was induced by 1ug/ml doxycycline.
Human lung fibroblasts IMR-90 cells were obtained from the Corielle Institute: NIA (National Institute on Aging) Aging Cell Repository. For generating procollagen stable HT-1080cell lines, we cloned proalpha(1) into a pRMc/CMV-vector and selected for neomycin resistance 39
. This vector was provided as a generous gift by Neil Bulleid. Cells were kept in a 37°C incubator at 5%CO2
siRNA screen in mouse ES cells
siRNA oligos against 40 mouse ubiquitin E3 enzymes were pre-designed by Qiagen and handled as instructed. Two different siRNA oligos against each gene were included in the initial screen. 10pmol of siRNA oligos and 0.25ul of Lipofectamine2000 were pre-incubated in a 0.1% gelatin-coated 96-well plate in 20ul of OPTIMEM for 15min at room temperature. The D3 mESCs were trypsinized and seeded at 15000 cells/well in 80ul of ESC medium on top of the siRNA mixture. Fresh medium was added to the cells the next day and the morphology of ES cell colonies were examined using bright-field microscopy at 48h post transfection. Hit validation was performed with additional siRNAs that were purchased from two distinct vendors (Qiagen, Dharmacon) and that target different sites of the Cul3 mRNA. Knockdown efficiency was tested by qRT-PCR and immunoblot.
Rescue of Cul3-siRNA phenotype in mESCs by matrigel and collagen-IV
D3 mESCs were cultured on tissue culture dishes coated with gelatin (negative control), growth-factor depleted matrigel (BD Biosciences, cat# 356231), or purified collagen-IV (BD Biosciences, cat# 354233). Matrigel and collagen-IV were applied at 10μg/cm2. Cul3 was depleted 24h later using our standard siRNA transfection protocol, and mESC morphology was analyzed by confocal microscopy against β1-integrin, actin, and DNA.
Drug treatments of Cul3-depleted cells
To study the synthetic lethal effect of Src-inhibition with Cul3 knockdown, we treated wildtype and Cul3-depleted D3 mESCs with 0, 25, 50, 100nM of dasatinib for 18h before the phenotypes were analyzed by light microscopy.
To study the effect of Rho-inhibition on Cul3 knockdown, Cul3-depleted D3 mESCs were treated with ROCK inhibitor Y27632 at 10uM for 24h before phenotype analysis. Alternatively, RhoA was co-depleted using specific siRNAs.
Cell cycle analysis
To assess the division rate of Cul3-depleted mESCs, we treated cells with control, Cul3-, or Ube2C/Ube2S-siRNA and seeded at 3×105
cells/well in gelatin-coated 6-well plates. The specificity of Ube2S- and Ube2C-siRNAs was tested before 40
. The cells were trypsinized at 2, 3 and 4d post transfection and counted by hemocytometer.
ES cell differentiation analysis
To differentiate mouse ES cells into embryoid bodies (EBs), we trypsinized undifferentiated D3 mouse ES cells, washed once with LIF-free ESC media, and seeded the cells at 2×106 cells/dish onto 10-cm Corning Ultra-Low-Attachment Dishes (Corning cat. # 3262) containing 10 ml of ESC medium without LIF. After 24h, the cells were dissociated from the plate by gentle pipetting of the medium and collected in a 15ml Falcon tube by centrifugation. The supernatant was aspirated off and the cells were re-seeded onto 10-cm Corning Ultra-Low-Attachment Dishes containing fresh ESC medium without LIF. Medium was changed every other day for a total of 6 or 9d. Total RNA of ESCs and EB samples was extracted using TRIzol (Invitrogen, cat. # 15596-026) and chloroform. The expression of pluripotent markers and BTB genes at various time points during differentiation was analyzed using quantitative real-time PCR.
As a complementary experiment, D3 mESCs were treated with control or Oct4 siRNA. 48h after transfection, cells were collected and total RNA was extracted using TRIzol as above. The expression of pluripotent markers, tissue specific genes and BTB genes in control and Oct4-depleted cells were analyzed using qRT-PCR.
Quantitative real-time PCR analysis
We used TRIzol (Invitrogen, cat. # 15596-026) and chloroform to extract total RNA from cells. The first-strand cDNAs were synthesized by using Revertaid first strand cDNA synthesis kit (Fermentas, cat. # K1621). Gene-specific primers for qRT-PCR were designed by using NCBI Primer-Blast. The quantitative RT-PCR reaction was done with the Maxima SYBR Green/Rox qPCR system (Fermentas, cat. # K0221).
Identification of Cul3Klhl12-substrates
To identify Cul3Klhl12 substrates, we generated a doxycycline-inducible hKlhl12-3xFLAG stable cell line using the Flp-In™ T-REx™ 293 Cell Line system (Invitrogen). As controls, we generated stable cell lines expressing other BTB proteins including Klhl9. Klhl12-3xFLAG and Klhl9-3xFLAG expression was induced in 30×15cm-plates by 1ug/ml of doxycycline for 48h, and cells were collected by centrifugation and lysed by douncing 40 times in PBS+0.1%NP40. The cell lysate was cleared by centrifugation and then subjected to anti-FLAG M2 affinity gel (Sigma, cat. # A2220-5mL) at 4C for 4h on a rotator. Immunoprecipitations were eluted by 300ul of 200ug/ml 3xFLAG peptide (Sigma, cat. # F4799-4MG) in PBS. The elution was repeated three times for 1h at room temperature. Eluates were pooled, concentrated to 100ul using Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore, cat. # UFC501008) and run on a SDS-PAGE gel. The gel was stained by SimplyBlue™ SafeStain (Invitrogen, cat. # LC6060), and specific gel bands were cut out and sent for mass spectrometry analysis by the Vincent J. Coates Proteomics/Mass Spectrometry Laboratory at UC Berkeley.
Immunoprecipitation of endogenous protein complexes
To confirm the interaction of endogenous proteins, we lysed Hela cells or D3 mESCs by freeze-thaw twice in 20mM HEPES buffer pH7.5, 5mM KCl, 1.5mM MgCl2, 1x protease inhibitor cocktail (Roche). Specific antibodies against Cul3, Sec13 or Sec31 conjugated to protein G agarose beads were added to the cleared cell lysate and incubated at 4C for 4h. Protein complexes were eluted with gel-loading buffer at 95°C. Endogenous proteins in complexes were detected by immunoblot using specific antibodies against Cul3, Sec13, Sec31, or Klhl12.
To detect ubiquitination of endogenous COPII components, we incubated Hela cell extract with pre-immune serum or antibody against Sec13 conjugated to protein G agarose beads at 4C for 4h. Protein complexes were eluted with SDS gel-loading buffer at 95°C. Ubiquitinated proteins in the complex were detected by immunoblot against ubiquitin.
In-vitro protein interaction assays
To dissect the Klhl12 and Sec31A interaction, we coupled 20ug recombinant MBPKlhl12, various mutants or MBP as a control to 15ul amylose resin by incubating at 4C for 1h. Cul3, Sec31A and mutants were expressed from pCS2 and labeled with 35S-Met using TnT Sp6 Quick Coupled Trsnc/trans Syst (Promega, cat. # L2080). The labeled Cul3 or Sec31A were incubated with MBP-Klhl12 or mutants at 4C for 3h. Beads were washed 4x with TBST and 2x with TBS, and incubated in SDS loading buffer at 95°C. Samples were run on SDS-PAGE and results were visualized by autoradiography.
In vitro ubiquitination assays with Cul3Klhl12
Cul3/Rbx1 was conjugated to NEDD8 at 30C for 1h with the following conditions: 2.5 mM Tris/HCl pH 7.5, 5 mM NaCl, 1 mM MgCl2, 1 mM DTT, 1x energy mix 40
, 1uM APPBP1-UBA3, 1.2 uM Ubc12, 4 uM Cul3/Rbx1, and 60 uM NEDD8. For in-vitro ubiquitination of Sec31A, we set up a 10ul reaction as follows: 2.5 mM Tris/HCl pH 7.5, 5 mM NaCl, 1 mM MgCl2, 1 mM DTT, 1x energy mix, 100nM UBA1, 1uM UbcH5c, 1uM Cul3~Nedd8/Rbx1, 1uM Klhl12, 150uM ubiquitin, 0.05ug Sec13/31A. The reaction was carried out at 30C for 1hr and stopped by adding SDS gel loading buffer.
In vivo ubiquitination assays with Cul3Klhl12
293T cells grown in 10cm dishes were transfected with pCS2-HA-Sec13/31A, pCS2-His-ubiquitin, pcDNA5-Klhl12-FLAG, pcDNA4-Cul3-FLAG, or pcDNA4-Cul3N250-FLAG, as indicated, using calcium phosphate. 24h later, 1μM MG132 was added and cells were incubated overnight. Cells were harvested with gentle scraping and resuspended in 1ml buffer A (6M guanidine chloride, 0.1 M Na2HPO4/NaH2PO4 and 10mM immidazole,pH 8.0). Cells were lysed by sonication for 10s and incubated with 25ul Ni-NTA agarose at room temperature for 3h. The beads were washed 2x with buffer A, 2x with buffer A/TI (1 volume buffer A and 3 volumes buffer TI), 1x with buffer TI (25mM Tris-Cl, 20mM imidazole, pH6.8), and incubated in 60ul SDS gel loading buffer containing 300mM imidazole and 50mM βME at 95°C. Samples were separated by SDS-PAGE and ubiquitinated Sec31A was detected by immunoblot using antibody against Sec31A.
To detect Sec31A ubiquitination upon Cul3/Klhl12 depletion, we co-transfected 100nM siRNAs against Cul3 or Klhl12 with pCS2-HA-Sec13/31A and pCS2-His-ubiquitin using calcium phosphate. The Ni-NTA purification was performed 48h post transfection and Sec31A ubiquitination was detected as described above.
Cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% TritonX-100 in 1X TBS, 2% BSA. Cells were incubated with primary antibodies against Sec31A, Sec13, Sec24C, ERGIC53, CD63, BiP, or ubiquitin for 2h and secondary antibodies (Invitrogen, Alexa Fluor® 546 goat anti-rabbit IgG (H+L); Alexa Fluor 488 goat anti-mouse IgG (H+L); HOECHST 33342,) for 1h at room temperature followed by extensive washing. Pictures were taken on Zeiss LSM 510 and 710 Confocal Microscope systems and analyzed with LSM image browser and Imaris 3D imaging processing software.
Transmission Electron Microscopy
Mock- and Klhl12-transfected HeLa cells were grown to 70% confluence as a monolayer on an Aclar ® sheet (Electron Microscopy Sciences, Hartfield, PA). The cells were fixed for 30min in 0.1M cacodylate buffer, pH 7.2, containing 2% glutaraldehyde, and subsequently washed with buffer prior to post-fixation with 1% Osmium tetroxide on ice. This was followed by staining with 1 % aqueous Uranyl Acetate for 30 min at room temperature. For dehydration with progressive lowering of temperature, each incubation period was 10 min, with exposure to 35% ethanol at 4°C, to 50% ethanol and 70% ethanol at −20°C, and 95%, and 100% ethanol at −35°C. Cells were restored to room temperature in 100% ethanol before flat embedding in an Epon resin. Thin (70-100nm) sections were collected on Formvar-coated 200-mesh copper grids and post-stained with 2% aqueous uranyl acetate and 2% tannic acid. The sections were imaged at 120 kV using a Tecnai 12 Transmission Electron Microscope (FEI, Eindhoven, Netherlands).
For the purpose of immunolabeling, HeLa cells expressing FLAGKlhl12 or doxycycline-inducible 293T Trex FLAGKlhl12 stable cell lines were fixed in 2% paraformaldehyde and 0.5% glutaraldehyde and embedded in LR white resin. Fixation and infiltration were performed in a microwave oven (Pelco model 3450, Ted Pella, Inc., Redding, CA). 70 nm thick sections were picked on 100-mesh nickel grids coated with Formvar film and carbon, incubated in blocking buffer (5% BSA, 0.1% fish gelatin, 0.05% Tween20 in PBS) for 30 min, and followed by incubation with αFLAG antibody at a dilution of 1/40 for 1h. Goat anti-mouse IgG conjugated with 10 nm gold (BD Biosciences) was used as the secondary antibody at a diltuion of 1/40 for 1 h. Sections were poststained in 2% uranyl acetate for 5 min.
Gene expression analysis by microarray
To compare gene expression profiles of WT-mESCs versus Cul3-depleted mESCs, we transfected D3 mESCs with control or Cul3-siRNA, followed by growth on gelatin-coated 6-well plates. 48h later, total RNA was extracted by TRIzol and chloroform, and further purified using RNeasy Mini Kit (Qiagen, cat. # 74104). Microarray analysis was performed by the Functional Genomics Laboratory (UC Berkeley) using Affymetrix Mouse 430A 2.0 chip.
Analysis of collagen export from cells
IMR-90 human lung fibroblasts grown on 100mm dishes in DMEM/10% FBS were transfected with FLAGKlhl12, FLAGKlhl12FG289AA, FLAGKeap1 and pcDNA5-flag using nucleofection kit R (bought from Lonza) as described in the manufacturer’s protocol and plated on 6 well plate with 25mm coverslips. When indicated, co-transfections with 2μg each of FLAGKlhl12 and dominant-negative Cul3 were performed. Dialyzed 10% FBS media was used for ascorbate free transfections. Brefeldin A (Sigma) was used at a concentration of 2.5mg/ml and cells were incubated for 30min. MG132 was used at 20μM for 2h, choloroquine was used at 200μM for 1h. Media was collected the next day and cells on coverslips were fixed with 3% paraformaldehyde for 30min and remaining cells on a plate were used to prepare lysates. Cells on coverslips were permebealized with 0.1% Triton for 15min at room temperature followed by blocking with 1%BSA for 30min. Primary antibodies used were polyclonal anti Procollagen (LF-67,diluted 1:1000) and anti-flag (diluted 1:200). Secondary antibodies were Alexa fluor 546 donkey anti-rabbit IgG and Alexa Fluor 488 goat ant-rabbit IgG (diluted 1:200). After staining cells with appropriate primary and secondary antibodies, we fixed coverslips on slides using mounting reagent containing DAPI. Images were analyzed with a Zeiss LSM710 confocal microscope and captured with Zen10 software. Merges of images were performed with ImageJ and LSM image Browser. Media collected from 6-well plates was normalized with respect to lysate protein concentration estimated using BCA method. Media and lysates of each reaction were checked by immunoblot analysis. Tubulin was used as loading control for lysates. Ascorbate chase experiments were done by adding ascorbate (0.25mM ascorbic acid and 1mM asc-2-phosphate) to Klhl12-transfected cells, followed by incubation for 5, 10, 30 and 60min.
A human fibrosarcoma cell line (HT1080) stably transfected with proalpha1(1) was used for Cul3 knockdowns. Cul3- and Klh12-shRNAs targeting two different regions in both genes were cloned into pSuperGFP and transfected using Lipofectamine 2000. pSuper GFP was used as negative control. Cells were grown on 25 mm coverslips in 6-well plates and fixed 2d post transfection. Collagen staining was done using LF-67 (1:1000) and ER was stained with anti-PDI (1:1000) antibody. Fibronectin and EGFR were stained in parallel experiments. Fibronectin expression was induced in HT1080 using 1uM dexamethasone before Cul3 knockdowns. ER retention or secretion was scored in cells expressing GFP shRNAs. Cells without GFP shRNAs and transfected with pSUPER GFP were quantified as well. Images were taken on a Zeiss LSM 710 confocal microscope and visualized with LSM image browser. Lysates were prepared from remaining cells on 6 well and checked for knockdown efficiency.
siRNA oligos used in this study
|RNAi oligos||Targeted sequence (5’ – 3’)|
|mSec13 (pool of 4 oligos)||CCATGTGTTTAGTAATTTA|
|mCdc42 (pool of 4 oligos)||GATCTAATTTGAAATATTA|
|mBcar1/CAS (pool of 4 oligos)||GACTAATAGTCTACATTTA|
|mSrc (pool of 4 oligos)||CCCTTGTGTCCATATTTAA|
|mRhoG (pool of 4 oligos)||GGTTTACCTAAGAGGCCAA|
|mRhoA (pool of 4 oligos)||GGATTTCCTAATACTGATA|
|mRac1 (pool of 4 oligos)||GGTTAATTTCTGTCAAACA|
|mCdh1/E-cadherin (pool of 4 oligos)||GGAGGAGAACGGTGGTCAA|
|mIqgap1 (pool of 4 oligos)||ACATGATGATGATAAACAA|
|mCul1 (pool of 2 oligos)||GCATGATCTCCAAGTTAAA|
|mCul2 (pool of 2 oligos)||GCGCTGATTTGAACAATAA|
|mCul4a (pool of 2 oligos)||GTGTGATTACCATAATAAA|
|mCul5 (pool of 2 oligos)||CCCTCATATTTACAGCAAA|
|mCul7 (pool of 2 oligos)||GCATCAAGTCCGTTAATAA|