Katayama et al., “FOXO transcription factor–dependent p15INK4b and p19INK4d expression”
Supplementary Materials and Methods = 711 words
1. Semiquantitative RT-PCR analysis: We extracted total RNA from cells with TRIZOL
reagent (Invitrogen), according to the manufacturer’s instructions. RT-PCR experiments
were carried out with cDNAs generated from 2 µg of total RNA using a GeneAmp RNA PCR
kit (Applied Biosystems, Foster City, CA). The RT-PCR exponential phase was determined
on 22-30 cycles to allow semiquantitative comparisons of cDNAs developed from identical
reactions with TaKaRa ExTaq polymerase (TaKaRa Bioscience, Kyoto, Japan). The primers
and PCR conditions are shown in supplementary Tables 1 and 2, respectively.
2. Cloning of pGL3 promoter vectors containing promoter region: The 1560-bp DNA
fragment of the p15INK4b promoter region and 975-bp DNA fragment of p19INK4d promoter
region containing a potential FOXO binding sequence were amplified by PCR with 293T
genomic DNA as a template. The 293T genomic DNA was extracted using a QIAamp DNA
mini kit (QIAGEN). The PCRs were performed using Pfu turbo DNA polymerase
(STRATAGENE) for p15INK4b and platinum Pfx DNA polymerase (Invitrogen) for p19INK4d
with the KpnI site-conjugated primers. The primer’s sequences and PCR conditions are
shown in supplementary Tables 3 and 4, respectively. The fragments were then digested by
KpnI and were cloned into a KpnI site of pGL3 promoter vector (Promega).
3. Cloning of pGL3 reporter vectors containing tandem copies of the putative
FOXO-responsive element: Double-stranded oligonucleotides containing a potential FOXO
binding sequence were generated by PCR with the single-stranded oligonucleotides
themselves as the template. The PCRs were performed using Pfu turbo DNA polymerase
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with the KpnI or BglII site-conjugated primers. The primer’s sequences and PCR conditions
are shown in supplementary Tables 5 and 6, respectively. The fragments were then
digested by both KpnI and BglII and were ligated into a KpnI-BglII sites of the pGL3
promoter vector. The number of tandem copies was determined by sequencing using an
ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).
4. Electronic mobility shift assay (EMSA): 293T cells were transfected with WT- or
HR-FOXO1a or WT- or HR-FOXO3a cDNA. Nuclear extractions were obtained using an
NE-PER extraction kit. Biotin end-labeled double-stranded oligonucleotides were
generated
by
annealing
the
5’-biotin-labeled
single-stranded
complementary
oligonucleotides containing FOXO consensus p15INK4b or p19INK4d gene sequences. The
sequences of the WT and mutated 21-bp double-stranded oligonucleotides to p15INK4b are
5’-biotin-GATAAATAAAAATAAGATACC-3’
(WT-p15INK4b)
and
5’-biotin-GATAACTAAACAGAAGATACC-3’ (Mut-p15INK4b), respectively. The sequences of
the WT and mutated biotin-labeled 18-bp double-stranded oligonucleotides to p19INK4d are
5’-biotin-AAAAACAAATCAGTTGCG-3’
(WT-p19INK4d)
and
5’-biotin-AAAACCAAAGCCGTTGCG-3’ (Mut-p19INK4d), respectively. Mutated nucleotides
are underlined. Non-labeled double-stranded oligonucleotides were also generated with the
non-labeled single-stranded complementary oligonucleotides using the same methods.
Binding reactions and electrophoresis have been described previously (Rokudai et al.,
2002), and detection of the biotin-labeled DNA was performed using a LightShift
chemiluminescent EMSA kit (Pierce), according to the manufacturer’s instructions.
5. Chromatin immunoprecipitation (ChIP) assay. 293T cells (2 X 106 cells) were transfected
with a pcDNA3 vector encoding none (Mock) or FLAG-tagged WT-, AAA-, or HR-FOXO1a.
After transfection for 24 h, we cross-linked the cells for 10 min by directly adding 1%
2
formaldehyde to the culture medium and then lysed them with lysis buffer (1% SDS, 10 mM
EDTA, 50 mM Tris-Cl, pH8.1). The lysates were sonified 6 times for 10 sec with output level
10 (Ultra5 homogenizer, Taitec, Saitama, Japan). For ChIP, the cell lysates were incubated
with an agarose-conjugated anti-FLAG M2 antibody (Sigma), and the following steps were
performed using EZ ChIP chromatin immunoprecipitation kit (Upstate, Charlottesville, VG).
The isolated DNA was amplified by PCR using an AmpliTaq Gold DNA polymerase (Applied
Biosystems) or an LA Taq DNA polymerase (TaKaRa). The primers and PCR conditions are
shown in supplementary Tables 7 and 8, respectively.
6. PI3K assay. Wild-type, INK4b-/-, and INK4d-/- MEFs were treated with or without 50 M
LY294002 for 24 h. The cells were then harvested and solubilized with lysis buffer as
described previously (Rokudai et al., 2002). The cell lysates (50 g) or recombinant PI3K
(1 g) (Cell Signaling Technology) were incubated with 1 M phosphatidylinositol (PI)
(Echelon Biosciences, Salt Lake City, UT) for 1 h in the presence of 1 mM ATP (TOYOBO,
Osaka, Japan). PI3K activity was determined using the QTL lightspeed class I
phosphoinositide 3-kinase (PI3K, PI3K, PI3K, PI3K) endpoint assay kit by measuring
fluorescence intensity (465 nm excitation, 595 nm emission).
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