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Materials and Methods
Small interfering RNA (siRNA) transfection
siRNA duplexes targeted against human HIF-1α as well as control scrambled siRNA were synthesized by Shanghai GenePharma Co., Ltd. The sense strands of the siRNA against HIF-1α were as follows: #1: CUGAUGACCAGCAACUUGATT, #2: AACUAACUGGACAVAGUGUGU.
Luciferase reporter assays
Osteosarcoma cells were seeded into 96-well plates and grown to 60% confluence before transfection. Cells were cotransfected with HIF-1α plasmid and plasmids HRE-luciferase-pGL4, P-luc-pGL4, PM1-luc-pGL4, PM2-luc-pGL4 or PM3-luc-pGL4 respectively, meanwhile Renilla luciferase was cotransfected as control of transfection efficiency. Luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega, Madison, Wis, USA).
Western blotting
Cells were harvested and lysed in 2X SDS gel loading buffer [24 mM Tris-HCl (pH 6.8), 0.02% mercaptoethanol, 4% SDS, 0.4% bromphenol blue, 20% glycerol]. Equal volumes of cell lysates were resolved on 8%–12% SDS-PAGE gels and transferred to PVDF membranes (Pierce Chemical). The blots were incubated for 1 h in a TBST solution supplemented with 5% non-fat dry milk，and with the appropriate indicated primary antibodies and then the suitable secondary antibodies conjugated with horseradish peroxidase. The primary antibody for HIF-1α was obtained from the BD Transduction Laboratories (San Jose, CA, USA)，WSB1, BNIP3, β-Actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA)，Myc-tag was obtained from Cell Signaling Technology (Beverly, MA, USA)and RhoGDI2 was from Spring Bioscience(Pleasanton, CA, USA). Secondary antibodies were from Santa Cruz Biotechnology.
Real-time PCR
Total RNA was extracted with EasyPure RNA Kit according manufacturer’s protocol (Transgen Biotech), and cDNA was synthesized with TranScript One-Step gDNA Removal and cDNA Systhesis SuperMix (Transgen Biotech). The relative mRNA levels were evaluated by quantitative RT-PCR using Eppendorfep Gradient Mastercycler (Eppendorf, Hamburg, Germany) and TAKARA SYBR Premix EXTaqTM. Primers used were as follows: GAPDH, forward primer: 5’-GTCATCCATGACAACTTTGG-3’and reverse primer: 5’-GAGCTTGACAAAGTGGTCGT-3’; WSB1, forward primer: 5’-CGTACTATAGGTGAACTTTTAGCTCCT-3’ and reverse primer:5’-CCAAAGGAAAACTGCTTTACTGG-3’.
Immunohistochemistry (IHC) assay
Breifly, the tissue sections were de-paraffinized and immersed for 10 min in PBS. For antigen retrieval, the sections were heated in microwave oven for 5 minutes in Citrate Antigen Retrieval solution and repeats 3 times. Endogenous peroxidase activity was blocked with 3% peroxide-methanol, and non-specific staining was then blocked with 10% goat serum. The sections were subsequently incubated with primary antibodies overnight at 4°C (WSB1 and RhoGDI2 were 1:100 diluted, the primary antibody for WSB1 was obtained from Proteintech Group and RhoGDI2 was from Spring Bioscience.) and treated for 20 minutes with biotinylated secondary antibody and HRP labled streptavidin respectively. The sections were then exposed for 6 min to 0.06% diaminobenzidine with 0.01% hydrogen peroxidase. The evaluation of the IHC staining was performed by pathologist. In brief, no staining (<5%) was scored as “-”, weak staining (5-25%) was scored as “+”, moderate staining (25-65%) was scored as “++” and strong staining (>65%) was scored as “+++”.
Chromatin immunoprecipitation (ChIP) assay
The primer sequences were as follows: 5’-CTCCCGGTTTCAAGCAATTC-3’ and 5’-TGCTAGGATTAGAGGCGTTAAG-3’ for PM1, 5’-CATGCCCTACAGTGTTATCATTTG-3’ and 5’-CAGTAGCTGGGATTTGTACAGG-3’ for PM2, 5’-CATATTAGACCATCCAGGCC-3’ and 5’-AGACCAAAGTACGGAGTCAA-3’ for PM3.
Immunofluorescence assay
Tumor hypoxia was assessed by injection of 30 mg/kg pimonidazole (PIMO) hydrochloride (Hypoxyprobe™-1 Kit, Chemicon) intraperitoneally 3h before mice scarified. Cryostat sections were fixed and permeated. PIMO, WSB1, HIF-1α antibodies (1:100 dilution) were used, followed by Alexa Fluor 488 or 594-conjugated and rhodamine secondary antibodies. Nuclei were visualized by staining with DAPI (Sigma Aldrich, D9542). Fluorescence signals were analyzed using an Olympus Fluorview 1000 confocal microscope (Olympus Corp. Melville, NY).
Immunoprecipitation of FLAG-WSB1 or HA-RhoGDI2
Cell lysate (250~400 µg from 293T cells) was incubated with 20 μl suspension of anti-FLAG or anti-HA beads for 1~2 h at 4 °C. After centrifugation at 500g for 2 min at 4 °C, the beads were washed three times with 500 μl washing buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 0.2% Nonidet P-40), and incubated with FLAG or HA peptide for 2h at 4 °C and the supernatant was used for later experiments.
Immunoprecipitation of endogenous RhoGDI2
Prot Elut NHS-Activated bead kit (Elut-P012, Enriching Biotechnology, Shanghai, China) was used for immunoprecipitation. Briefly, beads were coupled with anti-RhoGDI2 antibody, and then 500µg cell lysates was added in beads and incubated for overnight at 4℃ with end-over-end mixing. Finally, wash the complex with IP buffer for three times and add 30µl 0.1~0.2M glycine-HCl for 5 minutes at room temperature, then neutralize with 1M phosphate. The bound proteins were immunoblotted with EasyBlot anti-Rabbit or anti-Mouse IgG Kit (GeneTex, CA, USA).
Wound healing assay
Briefly,cells were seeded in 24-well plates until attached and form a monolayer on plates. We scraped the cells straightly using a pipette tip. After scraped, the cells were washed with PBS and were further incubated. The migration of cells across this artificial wound was assessed at different time intervals (0 and 24 hours) by microscopic observation.
Figure Legends
Figure S1. (A) Correlation between WSB1 expression levels and metastatic potential in osteosarcoma patients. (B) Schematic representation of full length WSB1 constructs with Myc-tag in lentivirus plasmid (pCCL). (C) The infection efficiency of different plasmid constructs in KHOS/NP cells validated by GFP expression. Scale bar, 200μm.
Figure S2. Wound healing assay of KHOS/NP cells infected with lentivirus-WSB1 or control lentivirus (pCCL). (A) Wound closure was monitored at the indicated time intervals and imaged. Scale bar, 200μm. (B) The wound area (scratch) was quantified. Bars represent mean±SD (n=3). **, p<0.01.
Figure S3. H&E staining of lung tissues in tail vein injection of KHOS/NP cells were performed in BALB/c (nu/nu) mice.
Figure S4. The discovery of downstream signaling of WSB1 via SILAC assay. A serial of metastasis-associated proteins identified in SILAC assay were quantified and shown as a histogram.
Figure S5. (A) Correlation between RhoGDI2 expression levels and metastatic potential in osteosarcoma patients. (B) Cells were exposed to hypoxia for 24 h or cells infected with control lentivirus (pCCL) and lentivirus-WSB1. RhoGDI2 mRNA expression was analyzed by RT-PCR and shown as a histogram after normalization, β-Actin was used as a control gene. (C) KHOS/NP cells treated with hypoxia for 24h together with 10 μM MG132, and RhoGDI2 protein levels were measured by western-blot analysis. (D) Colocalization of endogenous WSB1 and RhoGDI2. KHOS/NP cells were subjected to immunofluorescence staining with anti-WSB1 and anti-RhoGDI2 antibodies.
Figure S6. Time-lapse movie of representative KHOS/NP cells infected with control lentivirus (pCCL) on gelatin-coated surfaces.
Figure S7. Time-lapse movie of representative KHOS/NP cells infected with lentivirus-WSB1 on gelatin-coated surfaces.
Figure S8. Wound healing assay of KHOS/NP cells infected with lentivirus-WSB1, lentivirus-RhoGDI2, lentivirus-WSB1 plus lentivirus-RhoGDI2, or control lentivirus (pCCL). (A) Wound closure was monitored at the indicated time intervals and imaged. Scale bar, 200μm. (B) The wound area (scratch) was quantified. Bars represent mean±SD (n=3). **, p<0.01.
Table S1.The list of proteins with 3-fold change in WSB1 overexpression KHOS/NP cells using SILAC assay.