Cell culture and antibodies
Human non-small cell lung cancer (NSCLC) H460 and H1299 cells, and duodenal adenocarcinoma HuTu80 (previously named HTB-40) cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin, and maintained at 37 °C in humid air with 5% CO2 condition. Cell lines were tested routinely to confirm the absence of mycoplasma, and have been recently authenticated by short tandem repeat (STR) profiling (Bioresource Collection and Research Center (BCRC) of Food Industry Research and Development Institute, Taiwan). The hybridoma for Hermes-3 (H-3) anti-CD44 antibody was from the American Type Culture Collection (ATCC, Manassas, VA, USA). Antibodies against p-Src (Y416) (Cell Signaling Technology, Danvers, MA, USA), Src (Cell Signaling Technology), p-ERK1/2 (Cell Signaling Technology), ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-paxillin (Y118) (Biosource International, Camarillo, CA, USA), paxillin (Santa Cruz Biotechnology), FAK (BD Biosciences, San Jose, CA, USA) were used for immunoprecipitation and Western blot in this study. Unsaturated disaccharide standards of ∆CS-0S [∆UA-GalNAc], ∆CS-4S [∆UA-GalNAc(4S)], ∆CS-6S [∆UA-GalNAc(6S)], ∆CS-4S6S [∆UA-GalNAc(4S6S)], ∆HS-0S [∆UA-GlcNAc], ∆HS-6S [∆UA-GlcNAc(6S)], ∆HS-NS [∆UA-GlcNS], ∆HS-2SNS [∆UA(2S)-Glc(NS)], ∆HS-NS6S [∆UA-GlcNS(6S)], and ∆HS-tS [∆UA(2S)-GlcNS(6S)], where ∆UA is β-D-glucuronic acid, GalNAc is β-D-N-acetylgalactosamine, GlcNAc is α-D-N-acetylglucosamine GlcNS is α-D-N-sulfoglucosamine and S is sulfo) were purchased from Iduron (Macclesfield, UK).
Constructs and establishment of stable cell clones
SRGN cDNA was generated by polymerase chain reaction (PCR) using cDNA derived from 293 T cells as template and following SRGN primers (Forward 5′-CGA TGA ATT CAT GAT GCA GAA GCT ACT CAA ATG-3′ and Reverse 5′-CGA TGG ATC CTA ACA TAA AAT CCT CTT CTA ATC-3′). The PCR product was subcloned into p3xFLAG-CMV-13 vector (Sigma-Aldrich, St. Louis, MO, USA) using EcoRI and BamHI sites, followed by sequence confirmation. To generate SRGN(S/A) mutant cDNA, two-step PCR was performed using p3xFLAG-CMV-13-SRGN as template. In the first step, two reactions were performed using two sets of primers, respectively. Reaction 1 was performed using the SRGN forward primer and a reverse primer containing sequences corresponding to S/A mutation (5′-GCC AGC CCC AGC TCC TGC TCC AGC GCC GGC GCC GGC GCC GAA GCC TGC TCC AGC GTA GTC CTC AGA AAG TGG-3′) to generate SRGN(S/A) cDNA fragment 1. Reaction 2 was performed using forward primer containing sequences corresponding to S/A mutation (5′-TAC GCT GGA GCA GGC TTC GGC GCC GGC GCC GGC GCT GGA GCA GGA GCT GGG GCT GGC TTC CTA ACG GAA ATG-3′) and SRGN reverse primer to generated SRGN (S/A) cDNA fragment 2. In the 2nd-step PCR, the SRGN(S/A) cDNA fragments 1 and 2 were mixed and used as template, and PCR was performed using the SRGN forward and reverse primers to generate a full length SRGN (S/A) cDNA. The PCR product was subcloned into p3xFLAG-CMV-13 vector, followed by sequence confirmation. H1299 cells were transfected with p3xFLAG-CMV-13-SRGN and p3xFLAG-CMV-13 plasmids, respectively, by LF2000 (Invitrogen, Dublin, Ireland), and cultured in medium containing G418 (800 μg/ml, Sigma-Aldrich) for 14 days, yielding H1299/SRGN and H1299/SRGN(S/A) cell clones. The expression vector encoding the full-length standard form of CD44 was constructed by PCR amplification using human placenta cDNA as a template in the presence of CD44 primers (Forward 5′-AAG CTT GAC ACG ATG GAC AAG TTT TG-3′ and Reverse 5′-GAA TTC ATA ATG ATG TAG GTG TAA CAC-3′), followed by subcloning the PCR product into pcDNA 3.1/myc-His-A vector (Invitrogen) at XhoI and KpnI restricted sites, followed by sequence confirmation. To generate CD44-expressing clones, HuTu80 cells were transfected with CD44 expression vector using LF2000, and cultured in medium containing G418 (500 μg/ml) for 2 weeks. CD44-expressing HuTu80 cells were further sorted by flow cytometry using CD44-FITC antibody (clone G44–26, BD Biosciences). The resultant CD44-negative (HuTu80/Mock) and CD44-positive (HuTu80/CD44) cells were maintained in medium containing G418 (500 μg/ml). Knockdown of SRGN in H460 cells by lentivirus-shRNA has been previously described . SRGN-KD cells were maintained in medium containing puromycin (1 μg/ml, Sigma-Aldrich). CD44 was knocked out in H1299 cells using bacterial CRISPR/Cas9 system. In brief, CD44 sgRNA (5′-GCG CCA GGC TCA GCG GCA CG-3′), purchased from the National RNAi Core Facility, Taiwan, was constructed into the all-in-one sgRNA/CAS9 expression lentivector and Lentivirus-based knock-out of CD44 was performed following the online protocols (http://rnai.genmed.sinica.edu.tw/webContent/web/protocols) from the Core Unit. Single clones were selected and confirmed as CD44-null cell clones, and maintained in puromycin-containing medium.
Conditioned medium (CM) preparation and enzyme digestion of GAG chains
H1299/Mock, H1299/SRGN and H1299/SRGN (S/A) cells as well as H460/Sh-CTRL and H460/Sh-SRGN cells were seeded and cultured in 150-mm dishes. When cell density reaching 90% confluence, cells were washed with PBS twice and replenished with 20 ml of serum-free RPMI-1640 medium, followed by further incubation for 48 h. The CM was collected and filtered through 0.45 μm filter, concentrated, and desalted with ddH2O twice to a final volume of 100 μl by using Amicon Ultra-15 Centrifugal Filter Unit (UFC901024, EMD Millipore, Burlington, MA, USA) at 14,300×g centrifugation. Protein concentration in the concentrated CM was assessed by Bradford Protein Assay (BIO-RAD Life Science, Hercules, CA, USA). To digest SRGN GAG chains, an aliquot of CM that was measured to contain 75 μg protein was treated with 100 mU of Chondroitinase (Chase) B (Sigma-Aldrich), 100 mU of ChaseAC (Sigma-Aldrich), 100 mU of ChaseABC (Sigma-Aldrich), or 100 mU of Heparinase I + III (Sigma-Aldrich) for 24 h at 37 °C, followed by western blot analysis using designated antibodies, including anti-SRGN (HPA000759, Sigma-Aldrich), anti-HS (amsbio LLC, Cambridge, MA, USA), anti-∆HS stub (amsbio LLC), anti-CS (Abcam, Cambridge, UK), anti-∆C4S stub (Sigma-Aldrich) and anti-∆C6S stub (LifeSpan Biosciences, Seattle, WA, USA).
GAG purification and high performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of GAG disaccharide units
CM was prepared and concentrated as described above. Protein concentration was determined using Bradford reagent. For GAG purification, an aliquot of CM that was measured to contain 250 μg protein was mixed with 100 μl of actinase E (20 mg/ml), with ddH2O added to a final volume of 600 μl, and incubated at 55 °C for 24 h. After heat inactivation at 100 °C for 10 min, the reaction mixture was centrifuged at 10,000×g for 10 min at 4 °C. The supernatant was collected and pellet was re-suspended in 50 μl of ddH2O and centrifuged at 10,000×g for 10 min at 4 °C to collect the supernatant. The supernatants were combined, mixed with 200 μl of Urea buffer (8 M urea, 2% CHAPS, pH 8.3), and loaded onto a Vivapure MiniQ H spin column (#VS-1X01QH24, Sartorius Corporate, Goettingen, Germany) pre-equilibrated with the urea buffer. After spinning at 2000×g for 5 min at 4 °C, the flow-through was collected and re-loaded to the same column for spinning. These procedures were repeated for two more times. The column was washed by 400 μl of wash buffer (200 mM NaCl) by spinning at 2000×g for 5 min at 4 °C, and eluted by 400 μl of elution buffer (2.74 M NaCl) by spinning. The elution step was repeated for two more time. The eluents were combined (~ 1.2 ml) and concentrated to a volume of 50 μl by an Amicon Ultra-0.5 Centrifugal Filter Unit (#UFC500396, 3 kDa, Millipore) centrifuged at 14,300×g at 4 °C, and desalted by mixing with 450 μl of ddH2O followed by centrifugation for six times. The desalted GAGs sample was then treated with 100 mU of ChaseABC and 100 mU of Heparinase I + III for 24 h at 37 °C. The GAG samples were lyophilized and disaccharides were subjected to fluorescence labeling with 2-aminoacridone (AMAC). The freeze-dried disaccharides (2 μg) was added in 10 μl 2-aminoacridone (AMAC) solution (100 mM AMAC in glacial acetic acid/dimethyl sulfoxide (DMSO), 3:17 v/v) and incubated at room temperature for 15 min. Then, 10 μl of 1 M NaBH3CN was added to the reaction mixture and incubated at 45 °C for 4 h. The samples were lyophilized and ready for LC-MS/MS analysis . For standard curve, the AMAC-labeled disaccharides standards mixture (sodium salt of 10 unsaturated CS/DS disaccharides; Iduron, UK) was diluted to series concentrations (1–27 ng/ul) in 50% (v/v) aqueous DMSO.
LC-MS/MS analysis was performed by an ACQUITY UPLC I-Class System (Milford, MA, USA) coupled with a mass spectrometer (Orbitrap Elite, Thermo Scientific, Waltham, MA, USA) equipped with heated electrospray ionization (HESI) source. The derivatized samples are separated by gradient elution on a C18 column (BEH C18 column, 2.1 mm × 150 mm, 1.7 μm, Waters, Milford, MA, USA) at a flow rate of 200 μl/min. The column temperature was maintained at 25 °C. The mobile phases consisted of 80 mM ammonium acetate (eluent A) and 100% methanol (eluent B). The gradient program was linearly changed as follows: 12% B from 0 to 5 min, 12–14% B from 5 to 15 min, 14% B from 15 to 17.5 min, 14–30% B from 17.5 to 32.5 min, 30–100% B from 32.5 to 62.5 min. Mass spectra were acquired in Negative ion mode for MS and MS/MS function. Quantification analysis of AMAC-labeled disaccharides was performed based on Standard Curve method. The linearity is evaluated by the correlation coefficient (r2). The absolute amount of disaccharides in samples was calculated based on the equation of standard curve. Data acquisition and peak area integration were accomplished by using Xcalibur 2.2 software (Thermo Fisher Scientific). Standard calibration curves were generated with three technical replicates of five concentrations and used for determination of the absolute amount of the individual HS and CS disaccharides.
Cell migration assay and immunofluorescence of actin cytoskeleton reorganization
For Boyden chamber migration assay, 2 × 106 cells were seeded in 60-mm dish and incubated overnight. Cells were then incubated in serum-free medium for 24 h, and suspended by enzyme-free Gibco Cell Dissociation Buffer (Thermo Fisher Scientific), washed by complete cultured medium once, washed by PBS twice, re-suspended in RPMI medium containing 0.1% FBS, and subjected to migration assay using the standard 48-well chemotaxis chamber as previously described . Briefly, cells were seeded in the upper chamber (3 × 103 cells/well in 50 μl of medium containing 0.1% FBS), with lower chamber loaded with medium containing 5% FBS. In some experiments, the lower chamber was loaded with medium containing 2% FBS as specified. The lower surface of the membrane (#K80SH58050, GE Healthcare Life Sciences, Pittsburgh, PA, USA) inserted between the two chambers was coated with 0.1 μg/μl of fibronectin (Millipore). After 3 or 5 h as specified, the cells migrated through the insert and attached on the lower surface of the insert were stained with 1 μg/ml of Hoechst 33258, and counted by ImageJ. In some experiments, cells were re-suspended in the CM that was pretreated with or without ChaseABC and loaded into the upper chamber for migration assay. To inhibit Src activity, cells were incubated in serum-free medium for 24 h, and incubated in medium containing 10 μM of PP2 for 3 h, followed by Boyden chamber migration assay.
For wound healing assay, 1 × 104 cells were seeded into the Culture-Insert 2 Well (ibidi Gmbh, Grafelfing, Germany) and incubated overnight. Following cell attachment, inserts were removed and cells were washed with PBS twice. H1299 cells were cultured in serum-free RPMI medium and H460 cells were cultured in 2% FBS-containing RPMI medium for 72 h. Cell migration was observed under microscope at 24-h intervals. To observe F-actin cytoskeleton reorganization during the process of wound closuring, cells were seeded into the Culture-Insert 2 Well on slides, and allowed cells to migrate for a designated time (24 h for H1299 cells and 72 h for H460 cells), followed by cell fixation in 4% paraformaldehyde for 30 min. Cells were washed with Tris-Buffered Saline (TBS, 50 mM Tris-Cl, pH 7.6; 150 mM NaCl) three times and blocked with TBS containing 0.1% Triton X-100 and 3% bovine serum albumin (BSA) overnight at 4 °C. F-actin was stained by Alexa Fluor™ 594 Phalloidin (Invitrogen) and cells were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed under a laser-scanning confocal system (MRC 1000; Bio-Rad). Since overexpression and knockdown of SRGN had little effects on cell proliferation, the measurements of cell migration and wound closure were not corrected by cell proliferation.
Rho GTPase activation assay
5 × 106 cells were seeded in serum-free medium on FN-coated dishes and incubated for 30 min. Cells were lysed and assayed for Rac1 and Cdc42 activation using Rac/cdc42 Activation Assay Kit (Upstate Biotechnology, Lake Placid, NY, USA) and RhoA activation using RhoA Activation Assay (Upstate Biotechnology). Briefly, cells were lysed in 1X MLB lysis buffer containing 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM NaF and 1 mM Na3VO4. Two mg of cell lysate was mixed with 20 μg of PAK-1 PBD agarose and 0.3 mg of cell lysate was mixed with 50 μg of Rhotekin-RBD agarose, and the reaction mixtures were rocked gently at 4 °C for 60 min. Agarose was collected by centrifugation at 14,000×g for 5 s, and washed with 1X MLB three times. Agarose was resuspended in 20 μl of 2X Laemmli reducing sample buffer and boiled for 5 min. The supernatant and agarose pellet were mixed and subjected to Western blot analysis. Rac1-GTP, Cdc42-GTP and RhoA-GTP pulled down by agarose beads were detected using specific antibodies.
CD44-Fc pulldown assay
Cells were seeded onto four 150-mm dishes and CM was prepared after cells were cultured in serum-free medium for 48 h as described above. The CM was concentrated and mixed with 1 μg of human CD44-Fc recombinant fusion protein (R&D Systems, Minneapolis, MN, USA) or IgG control and rocked gently in a rotating shaker at 37 °C for 18 h. The reaction mixture was mixed with 20 μl of magnetic beads reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA,) and incubated at 37 °C for 2 h. Magnetic beads were collected by magnetic stand and supernatant discarded. The beads were washed with wash buffer (25 mM Tris-HCl, pH 7.6, 200 mM NaCl, 0.1% NP-40, 6% glycerol, 1 mM EDTA and 0.5 mM DTT) for three times, and suspended in 20 μl of 2X Laemmli reducing sample buffer and boiled for 5 min, followed by western blot analysis using anti-SRGN antibody.
Data were presented as mean ± s.d. from at least three independent experiments. Two-tailed Student’s t test was employed. P < 0.05 was considered to be statistically significant. *, P < 0.05; **, P < 0.01 and ***, P < 0.001.