Supplementary methods
1. Construction of Luciferase report vectors
The procedures have been described in Chen et al (Chen et al., 2011). Briefly, DNA
fragments were PCR amplified from human genomic DNA and cloned into the multiple
cloning site (XhoI and NotI) distal to the Renilla luciferase coding region of the psiCHECK2
vector (Promega, Madison, WI). PCR was performed using KOD hot start DNA polymerase
(Novagen, Madison, WI). The primer sequences used to construct vector psiCHECK-STMN1
contains wild type STMN1 3’ UTR were as follows:
XhoI-STMN1-FW
5’ - ACGCCTCGAGTTGTTCTGAGAACTGACTTTCTCC - 3’
NotI-STMN1-RV
5’ - ATAAGAATGCGGCCGCATATTCTGATTCTCGTGTCATAGC - 3’
Overlapping PCR was used to generate vector psiCHECK-STMN1 M, which contains a
deletion at the miR-193b seed binding site. Two additional primers were used:
STMN1-d58-64-FW: 5’ - ATATCCAAAGACTGTACTTCATTTTATTTTTTCCCTG - 3’
STMN1-d58-64-RV: 5’ - AGTACAGTCTTTGGATAT - 3’
2. Construction of STMN1 expression lentiviral vector
Full-length STMN1 cDNA (NM_005563.3) was PCR amplified from a STMN1 TrueClone
plasmid SC116654 (OriGene Technologies, Rockville, MD), and was subsequently
subcloned into the PmeI site of WPI lentiviral vector as described in Hao et al (Hao et al.,
2008). Two primers used to PCR STMN cDNA:
PmeI-STMN1-F: 5’-AGCTTTGTTTAAACCATGGCTTCTTCTGATATCCAG-3’
PmeI-STMN1-HA-R: 5’-AGCTTTGTTTAAACATTACGCGTAGTCTGGGACATCGTATGGATA
GTCAGCTTCAGTCTCGTCAGCAGG-3’
PmeI site is underlined. Start codon and stop codon are in italic. HA tag is in bold.
3. Gene expression analysis.
Malme-3M cells were seeded 600,000 cells per plate in 100-mm dishes and transfected the
following day with 10 nmol/L either negative control siRNA or STMN1 siRNA. Total RNA
was harvested 24 hours after transfection using miRNeasy Mini kit (Qiagen). The Agilent
Microarray Platform for One-Color Analysis of Gene Expression was used for profiling
(Whole Human Gene Expression Microarray, 4x44K Agilent). The detailed procedure of
microarray was described in our previous study (Chen et al., 2010). Raw data were first
normalized and analyzed using GeneSpring GX 11.5.1 (Agilent). For quality control, the
probes must be present in one out of two conditions and flagged as “Dectected.” P values
were calculated for two pair-wise comparisons between Malme-3M cells with or without
STMN1 knockdown using the paired t test. A total of 336 entities (genes or transcripts)
were identified with at least 1.5 fold change, including 215 downregulated entities and 121
upregulated entities. After filtering multiple entities mapped to a single gene and
unmapped entities, ingenuity pathway analysis (IPA) identified 170 downregulated genes
and 102 upregulated genes (Supplementary Table 1). Core analysis in IPA was performed
subsequently.
References
Chen J, Feilotter HE, Pare GC, Zhang X, Pemberton JG, Garady C et al. (2010). MicroRNA193b represses cell proliferation and regulates cyclin D1 in melanoma. Am J Pathol 176:
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Chen J, Zhang X, Lentz C, Abi-Daoud M, Pare GC, Yang X et al. (2011). miR-193b regulates
mcl-1 in melanoma. Am J Pathol .
Hao Y, Chun A, Cheung K, Rashidi B, Yang X. (2008). Tumor suppressor LATS1 is a negative
regulator of oncogene YAP. J Biol Chem 283: 5496-5509.