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Supplementary Materials and Methods
Small RNA isolation and library generation
Total RNA (5-50 μg/sample) was size-fractionated by denaturing PAGE and adapter
oligonucleotides were ligated at both ends of the small RNA molecules. After reverse transcription
(Invitrogen), cDNA was PCR-amplified twice. A PmeI digestion (New England Biolabs) was
performed in between the two rounds of PCR to degrade the 19- and 24-mer used during the size
fractionation procedure. Finally, the purified PCR products were quality controlled by Agilent 2100
Bioanalyzer (Agilent Technologies) to assess acceptable and comparable features and then
sequenced using 454 technology (454 Life Science) at the Memorial Sloan Kettering Cancer Center,
thanks to Agnes Viale’s help. To minimize batch-dependent effects, all primary samples (both
muscle and tumors) were processed in parallel from RNA extraction throughout the whole library
preparation procedure. Primer sequences are listed below.
Lentiviral vectors and siRNAs
Conditional NpBI-22 and NpBI-378 lentiviral vectors were generated by PCR amplification,
respectively, of the pre–miR-22 and pre-miR-378 loci from human genomic DNA. The PCR
products were cloned into the SacII and KpnI sites of pCCL.sin.PPT.hPGK.GFPWpre vector
provided by Luigi Naldini (San Raffaele-Telethon Institute for Gene Therapy, Milano, Italy),
previously described (1). Conditional NpBI lentiviral vectors were generated by subcloning the
bidirectional tetracycline-responsive element–GFP cassette from pBI vector (Clontech) into
pCCL.sin.PPT.hPGK.GFPWpre-miR-22 . Concentrated lentiviral vector stocks were produced as
previously described (2). To obtain regulatable expression of miRNAs, cells were transduced first
with a lentiviral vector expressing the reverse tetracycline transactivator (rtTA) (Tet-on system) and
subsequently with the responder vector. To promote inducible vector expression, cells were treated
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with 1 µg/ml of doxycycline for the indicated times. NpBI-22 Delta_miR vector was generated
from NpBI-22 vector according to Agilent Technologies Mutagenesis kit protocol. Human TACC1
CDS was purchased from Open Biosystems/Dharmacon, sequence validated and subcloned into the
BamHI/SalI restriction sites of the pCCL.sin.PPT.hPGK.GFPWpre vector. Human RAB5B CDS
was PCR amplified from genomic DNA, sequence validated and sub-cloned into the BamHI/SalI
restriction sites of the pCCL.sin.PPT.hPGK.GFPWpre vector. PLKO.1 lentiviral vectors expressing
shRNA against TACC1 (code: TRCN0000275917) and the control shRNA (code: SHC002) were
purchased from Sigma-Aldrich. Isoform-specific siRNAs against RAB5A, B and C were kindly
provided by Dr. Letizia Lanzetti and sequences are available on request. Negative Control siRNA
from ThermoFisher (Cat#4611G) was used as a control. Transient transfections were performed
with Lipofectamine 2000 (Invitrogen) following standard procedures. Oligonucleotides used for
miRNAs and RAB5B amplification are listed below.
Sensor vector generation and reporter assays
The GFP-TACC1 wild type or mutated sensor vectors (containing two repetitions of the two miR22 MREs) were obtained by annealing oligonucleotides listed below. Annealed oligonucleotides
were then subcloned into the SacII/KpnI sites of the aforementioned lentiviral vector. GFP sensor
vectors and 50nM synthetic pre-miRNA (miRNA precursor hsa-miR-22, PM10203 ThermoFisher)
were co-transfected in HEK 293T cells with Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s instructions. Cells were harvested after 48 hours and green fluorescence was
measured by flow cytometry using CellQuest Software. The Luciferase-RAB5B sensor vectors
(containing the three miR-22 MREs) were obtained by annealing oligonucleotides listed below.
Primers #1 contain the first two MREs (wild type or mutated), while primers #2 contain the last
MRE (wild type or mutated). The Luciferase-ERBB3 sensor vectors (containing the miR-22 MRE)
were obtained by annealing oligonucleotides listed below. For both RAB5B and ERBB3, annealed
oligonucleotides were subcloned into psiCHECKTM-2 vector (Promega) downstream of Renilla
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luciferase using the XhoI/NotI restriction sites. The psiCHECKTM-2 vectors were co-transfected
with 50 nM synthetic pre-miRNA (miR-22, see details above) in HEK 293T cells with
Lipofectamine 2000 (Invitrogen) and the luciferase assay was performed with the Dual-Luciferase
Reporter Assay System (Promega). Luminescence was measured with the ‘Dual Glo’ protocol of
the GloMax Multi Detection System (Promega) 48 hours post-transfection. The luciferase signal
obtained from Renilla was normalized against Firefly luciferase intraplasmid control. Pre-miR-22
promoter fragments were generated by PCR using murine genomic DNA as a template. Amplified
fragments were digested with EcoRV/HindIII, cloned into pGL4-basic luciferase reporter vector
(Promega) and sequenced. Mutant constructs were made by site-directed mutagenesis using the
QuikChange Mutagenesis Kit (Stratagene). Oligonucleotides used are listed below. All mutant
constructs were verified by sequencing. Vectors were cotransfected into NIH 10T½ NpBI-MyoD
cells along with 1:10 of pRL-TK Renilla control vector using Lipofectamine 2000. 12 hours after
transfection, fresh medium with or without doxycycline was added. 48 hours later, cells were lysed
and assayed for Luciferase and Renilla expression as described above. Renilla values were used to
normalize for differences caused by unequal transfection efficiency.
Scratch wound assay
Cells were plated at sub-confluence. For rescue experiments, cells were plated in the presence of
doxycycline and wounds were made 24h after plating. For loss of function experiments, wounds
were made 24h after transfection. Several wounds were made across the confluent cell monolayer in
replicate wells using a standard 1ml pipette tip, then cells were washed twice with PBS and
incubated in DMEM 10% FBS. Wound areas were calculated with ImageJ from multiple pictures
(5X magnification) taken for each condition at the indicated times. Percentage of wound closure
was calculated as previously described (3).
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Oligonucleotides
Oligonucleotides used for miRNA library generation
Adapter oligonucleotides:
3’_adapter: 5’-TCGTATGCCGTCTTCTGCTTG-3’;
5’_adapter: 5’-GUUCAGAGUUCUACAGUCCGACGAUC-3’.
PCR primers:
st
1 _PCR_for: 5’-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3’;
1st_PCR_rev: 5’-CAAGCAGAAGACGGCATACGA-3’;
2nd_PCR_for: 5’-GCCTCCCTCGCGCCATCAGAATGATACGGCGA-3’;
nd
2 _PCR_rev: 5’-GCCTTGCCAGCCCGCTCAGCAAGCAGAAGACG-3’.
PmeI digestion oligonucleotides:
19-mer: 5’-CGUACGCGGGUUUAAACGA-3’;
24-mer: 5’-CGUACGCGGAAUAGUUUAAACUGU-3’.
Oligonucleotides used for Northern blot
anti–miR-22: 5′-ACAGTTCTTCAACTGGCAGCTT-3′;
anti–miR-378: 5′-CCTTCTGACTCCAAGTCCAGT-3′;
anti-U6: 5′-TGTGCTGCCGAAGCGAGCAC-3′.
Oligonucleotides used for lentiviral vectors production
Oligos for NpBI-22 and NpBI-378 lentiviral vectors:
pre–miR-22_for: 5’-CAGCGAGGTTAACAGCTTCCGCGG-3’;
pre–miR-22_rev: 5′-CTGGTACCGGTGATGGTATCAGGGATGG-3′;
pre–miR-378_for: 5′-TGCCGCGGGCGAGCCTAGCTAGCAGAAA-3′;
pre–miR-378_rev: 5′-CTGGTACCTCACATGCAAACACTGCTCA-3′.
Oligos for NpBI-22 Delta_miR lentiviral vector:
Delta_miR_for: 5’-GACCCAGCTAAAGGACACAGTTGAAGAACTG-3’;
Delta_miR_rev: 5’-CAGTTCTTCAACTGTGTCCTTTAGCTGGGTC-3’.
Oligos for RAB5B lentiviral vector:
RAB5B_for: 5’-ggatccATGACTAGCAGAAGCACAGCTA-3’;
RAB5B_rev: 5’-gtcgacTCAGTTGCTACAACACTGGCTC-3’.
Oligonucleotides used for sensor vectors generation
TACC1_WT_for1: 5’-GGAAAAGGAAAGTAGAAGGCAGCTGCCGATAAAAGGAAAGTAGAAGGCAGCTGCA-3’;
TACC1_WT_for2: 5’-AGCTTATAAAGGTAACAAAGGGCAGCTCACGATATAAAGGTAACAAAGGGCAGCTCAGGTAC-3’;
TACC1_WT_rev1: 5’-AGCTTGCAGCTGCCTTCTACTTTCCTTTTATCGGCAGCTGCCTTCTACTTTCCTTTTCCGC-3’;
TACC1_WT_rev2: 5’-CTGAGCTGCCCTTTGTTACCTTTATATCGTGAGCTGCCCTTTGTTACCTTTATA-3’;
TACC1_MUT_for1: 5’-GGAAAAGGAAAGTAGAACCGTCGACCCGATAAAAGGAAAGTAGAACCGTCGACCA-3’;
TACC1_MUT_for2: 5’-AGCTTATAAAGGTAACAAAGCCGTCGAGACGATATAAAGGTAACAAAGCCGTCGAGAGGTAC-3’;
TACC1_MUT_rev1: 5’-AGCTTGGTCGACGGTTCTACTTTCCTTTTATCGGGTCGACGGTTCTACTTTCCTTTTCCGC-3’;
TACC1_MUT_rev2: 5’-CTCTCGACGGCTTTGTTACCTTTATATCGTCTCGACGGCTTTGTTACCTTTATA-3’;
RAB5B_WT_for1: 5’-TCGAGTTCAGCAACAAACACCAGGCAGCTGTTCATATGTAGATAAAACTACCAGTGGCAGCTACTA-3’;
RAB5B_WT_for2: 5’-AGCTTTGAAGTTCTTTCTCTGTGCAGCTTTCCGC-3’;
RAB5B_WT_rev1: 5’-AGCTTAGTAGCTGCCACTGGTAGTTTTATCTACATATGAACAGCTGCCTGGTGTTTGTTGCTGAAC-3’;
RAB5B_WT_rev2: 5’-GGCCGCGGAAAGCTGCACAGAGAAAGAACTTCAA-3’;
RAB5B_MUT_for1: 5’- TCGAGTTCAGCAACAAACACCATTAGTACGTTCATATGTAGATAAAACTACCAGTTTAGTACACTA-3’;
RAB5B_MUT_for2: 5’-AGCTTTGAAGTTCTTTCTCTGTTAGTACTTCCGC-3’;
RAB5B_MUT_rev1: 5’-AGCTTAGTGTACTAAACTGGTAGTTTTATCTACATATGAACGTACTAATGGTGTTTGTTGCTGAAC-3’;
RAB5B_MUT_rev2: 5’-GGCCGCGGAAGTACTAACAGAGAAAGAACTTCAA-3’;
ERBB3_WT_for: 5’-TCGAGAGATCTCAGGGAGCATTTAATGGCAGCTAGC-3’;
ERBB3_WT_rev: 5’-GGCCGCTAGCTGCCATTAAATGCTCCCTGAGATCTC-3’;
ERBB3_MUT_for: 5’-TCGAGAGATCTCAGGGAGCAGAACCGTTACTAGGGC-3’;
ERBB3_MUT_rev: 5’-GGCCGCCCTAGTAACGGTTCTGCTCCCTGAGATCTC-3’;
22.0_for: 5’-TGGATATCTGTCCTCCCTCAGACTCCTG-3’;
22.1_for: 5’-TGGATATCGGCTTGGAACTTGGAACAAA-3’;
22.2_for: 5’-TGGATATCAACTCATTTGGCCTGTCACC-3’;
22_rev: 5’-CAAAGCTTTCCTAAAAGGAAGGGGAGGA-3’;
22.1 I mut_for: 5’-GCCTGGAATTCATACtgGAcaTACCCGGAATGTGC-3’;
22.1 I mut_rev: 5’-GCACATTCCGGGTAtgTCcaGTATGAATTCCAGGC-3’;
22.1 II mut_for: 5’-CTCCTGCCTTTGGTtgGCcaTCCTCCCTCAGACTC-3’;
22.1 II mut_rev: 5’-GAGTCTGAGGGAGGAtgGCcaACCAAAGGCAGGAG-3’.
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Oligonucleotides used for real-time PCR analysis
RAB5A_for: 5’-CAACGGGCCAAATACGGGAAAT-3’;
RAB5A_rev: 5’-CAGCCCCAATGGTACTCTCTTGAA-3’;
RAB5B_for: 5’-GACTAGCAGAAGCACAGCTAGG-3’;
RAB5B_rev: 5’-GAACTGCCCTTTGACAAAACGTAAT-3’;
RAB5C_for: 5’-GGAACTGAGTTGGAGGTCCCC-3’;
RAB5C_rev: 5’-TTGACAGATCTTGTTCCCAGCAGC-3’;
HuPO_for: 5’-GCTTCCTGGAGGGTGTCC-3’;
HuPO_rev: 5’-GGACTCGTTTGTACCCGTTG-3’.
Oligonucleotides used for ChIP assay
MCK_enhancer_for: 5’-AGGGATGAGAGCAGCCACTA-3’;
MCK_enhancer_rev: 5’-CAGCCACATGTCTGGGTTAAT-3’;
IgH_enhancer_for:5’-GCCGATCAGAACCAGAACACCTGC-3’;
IgH_enhancer_rev: 5’-TGGTGGGGCTGGACAGAGTGTTTC-3’;
MiR-22_oligoA_for: 5’-ATGTACCCGGAATGTGCAAG-3’;
MiR-22_oligoA_rev: 5’-CAGGAGTCTGAGGGAGGACA-3’;
MiR-22_oligoB_for: 5’-GGGCAGAGAGAAGTGGACAG-3’;
MiR-22_oligoB_rev: 5’-CTAGGGTGGGCAACTTTCAA-3’.
Antibodies
Antibodies for FACS
Anti-MHC
Hybridoma Bank (University of Iowa)
Alexa-555
Invitrogen
Antibodies for Western blot
Anti-MHC
Santa Cruz Biotechnology
Anti-RAB5B (A-20)
Santa Cruz Biotechnology
Anti-Actin
Sigma-Aldrich
Anti-TACC1
Sigma-Aldrich
Anti-P-MAPK
Sigma-Aldrich
Anti-GAPDH
Cell Signaling Technology
Anti-P-EGFR (Tyr-1068) Cell Signaling Technology
Anti-P-ERBB3 (Tyr-1197) Cell Signaling Technology
Anti-P-Akt (Ser-473)
Cell Signaling Technology
Anti-ERBB3
Millipore
Anti-P-RB (Ser780)
Cell Signaling Technology
Anti-Cyclin D3
Santa Cruz Biotechnology
Anti-Cyclin A
Santa Cruz Biotechnology
Anti-cleaved Caspase-3 Cell Signaling Technology
Anti-HSP90
Cell Signaling Technology
Antibodies for immunohistochemistry
Anti-Ki67
Dako
Anti-cleaved Caspase-3 Cell Signaling Technology
Anti-Myogenin
Hybridoma Bank, University of Iowa
Antibodies for ChIP
Anti-MyoD (M-318)
Santa Cruz Biotechnology
Anti-acetyl-histone H3
Millipore
Rabbit IgG
Cell Signaling Technology
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Supplementary References
1. Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L. Gene transfer by lentiviral vectors is
limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet.
2000;25(2):217–22.
2. Taulli R, Scuoppo C, Bersani F, Accornero P, Forni PE, Miretti S, et al. Validation of met as a
therapeutic
target
in
alveolar
and
embryonal
rhabdomyosarcoma.
Cancer
Res.
2006;66(9):4742–9.
3. Chen P-I, Schauer K, Kong C, Harding AR, Goud B, Stahl PD. Rab5 isoforms orchestrate a
“division of labor” in the endocytic network; Rab5C modulates Rac-mediated cell motility.
PloS One. 2014;9(2):e90384.
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