Download Materials and Methods

Supplementary information for Liu et al., “Oncogenic BRAF regulates -Trcp
expression and NF-B activity in human melanoma cells”
Materials and Methods
Plasmids and chemicals Constructs for mammalian expression of Flag-tagged IB (Scherer
et al., 1995), and luciferase reporters driven by B- (DiDonato et al., 1996) and Trcp2
promoters (Spiegelman et al., 2002a) were previously described. Conditionally active forms of
BRAF-V600E (BRAFVE) were generated by fusion to modified forms of the hormone binding
domain of the human estrogen receptor (hbER, (Littlewood et al., 1995)). To facilitate the
generation of such fusion constructs a full length human BRAF cDNA encoding a 766 amino
acid form of human BRAFVE was subjected to PCR using the following primers;
BRAF1: 5’-CACCGTCGACACCATGGCGGCGCTGAGCGGTGGC-3’
BRAF2: 5’-CCCTAGTCGACTCACTACCTAGGCCCGTGGACAGGAAACGCACC-3’
These primers introduce a unique SalI site upstream of the codon for the initiator methionine
and a unique AvrII site between the final codon of BRAF (His766) and the normal stop codon
of the mRNA. This PCR product was cloned into a TOPO PCR fragment cloning vector.
Next sequences encoding the T2 form of the hormone binding domain of the human
estrogen receptor- [hbERT2] were subjected to PCR using the following PCR primers.
HBER1: 5’-CACCGGATCCACCATGGGGAACTAGGTCCGATCCATCTGCTCCAG-3’
HBER2: 5’-CCCCTAGTCGACTGTGGCAGGGAAACCCTCTGCCTCCCCCG-3’
These primers introduce a unique AvrII site upstream and a unique SalI site downstream of the
sequences encoding hbER.
-TOPO
cloning vector. Sequences encoding PCR amplified BRAFVE were fused in frame to sequences
encoding hbERT2 by subcloning a NotI-AvrII fragment of BRAFVE into the appropriate sites of
pENTR/D-TOPO:hbERT2 to generate pENTR vectors encoding BRAFVE:ERT2, in which ERT2
is fused to the C-terminus of activated BRAF.
Retrovirus vectors for expression of BRAFVE:ERT2 in mammalian cells were generated by
Gateway mediated subcloning into a variety of Gateway compatible retrovirus vectors. Mouse
Melan-a cells expressing BRAFVE:ERT2 were generated by infection with a retrovirus vector
encoding BRAFVE:ERT2 and resistance to Puromycin (gLXSP3). In this vector expression of
BRAFVE:ERT2 is promoted by the MuLV LTR and resistance to puromycin by the SV40 early
promoter. Retrovirus infected cells were isolated by selection in Puromycin for 7-10 days.
Activity of BRAFVE:ERT2 was induced by adding 4-hydroxytamoxifen (4-OHT, 250nM) to the
growth medium for 16hr to allow a robust induction of -Trcp.
MEK inhibitor PD 098059 and U0126 (Calbiochem), 4-hydroxytamoxifen (4-OHT)
and cycloheximide (Sigma), as well as tumor necrosis factor alpha (TNF, R&D
Systems) were purchased. BAY 43-9006 was a kind gift from Dr. Charles Smith
(Hershey Medical Center, Pennsylvania State University).
Cells Normal human melanocytes and human melanoma cells were cultured as
previously described (Satyamoorthy et al., 2003). Melan-A mouse melanocytes (Bennett
et al., 1987) or their derivatives that were engineered to express BRAFVE:ERT2 were
cultured in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, 200nM
tetradecanoyl phorbol-13-acetate, glutamine, penicillin and streptomycin. Cells were
transfected using Lipofectamine Plus (Invitrogen) following the manufacturer’s
recommendations. Luciferase assays in melanoma cells transfected with either B- or Trcp2 promotor-driven luciferase reporter constructs and renilla luciferase plasmid were
performed using Dual Luciferase kit (Promega). For RNAi approach, the shRNA that
specifically target V600E mutant allele of BRAF were described before (Hingorani et al.,
2003); cells were harvested 24hr after transfection with these shRNA plasmids.
The rate of apoptosis was measured by flow cytometric analysis upon the cell
staining with allophycocyanin conjugate (APC) labeled-Annexin V with or without 7Amino-actinomycin D (7-AAD) using Annexin V kit (BD Biosciences Pharmingen).
Immunotechniques Monoclonal antibodies against phospho-ERK1/2 or total
ERK1/2 (Cell Signaling), Flag tag (M2, Sigma), IKK (PharMingen), BRAF, IKK and
IB (Santa Cruz), as well as -actin and -tubulin (Sigma) were purchased. Polyclonal
antibody against -Trcp2 (HOS-N) was described earlier (Spiegelman et al., 2002b). An
IKK immunokinase assay was carried out with IKK antibody or Flag antibody as
described elsewhere (Dejardin et al., 2002). Immunoprecipitation and immunoblotting
procedures were described earlier (Spiegelman et al., 2002a).
References for Supplementary Information:
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