Measurement of cell proliferation
Cell proliferation was assessed with the cell counting kit-8 (CCK-8, Dojindo
Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. All of the
experiments were performed in triplicate. The cell proliferation curves were plotted
with the absorbance at each time point. In regard to the EdU immunofluorescence
staining, the 3×105 cells per well were seeded in cell culture 12-well plate with a
cover slip in every well. After growth to approximately 50% cell confluency cultures
were then assessed with an EdU immunofluorescence kit (RiboBio, Guangzhou,
China) according to the manufacturer’s protocol.
Reverse transcription reactions and quantitative real-time PCR
Total RNAs were extracted with Trizol reagent (Takara, Dalian, China). The
first-strand cDNA was generated with the Reverse Transcription System Kit (Promega
Corporation, Madison, WI). Real-time PCR was performed according to a standard
SYBR-Green PCR kit protocol in a StepOne Plus system (Applied Biosystems, Foster
City, CA). GAPDH was used as an endogenous control to normalize the amount of
total mRNA in each sample. All of the real-time PCR reactions were performed in
triplicate. The relative expression of RNAs was calculated with the comparative ⊿⊿
Ct method. The primer sequences are presented in Supporting Table 1. In regard to the
detection of miR-675, real-time PCR was performed with a Hairpin-itTM miRNAs
qPCR Quantitation kit purchased from Gene Pharma (Shanghai, China). For absolute
quantification of miRNA levels, threshold cycle (Ct) values were compared to the Ct
values of a standard curve produced by serial dilution and amplification of the
miRNA qRT-PCR Standard RNA(RIBOBIO). For absolute quantification of H19
levels, standard curves were created by real-time PCR using a dilution series of
pcDNA3.1-H19 vector.
Luciferase reporter assay
Approximately 500 bp of the promoter region of several hnRNP U-binding
genes were PCR-amplified by TaKaRa LA Taq (Takara) and subcloned into the pGL3
basic firefly luciferase reporter plasmid. The pGL3 basic promoter plasmid that
contained the promoter of the indicated genes was cotransfected along with the m77
vector, m77-hnRNP U(obtained from Genecopoeia, Guangzhou，China)，pcDNA3.1
or pcDNA-H19 into BNL CL.2 cells by Lipofectamine-mediated gene transfer. Each
sample was cotransfected with the pRL-TK plasmid, which expressed Renilla
luciferase, in order to monitor the transfection efficiency (Promega). The relative
luciferase activity was normalized to the Renilla luciferase activity. The TCF reporter
plasmid kit with the TOPFlash and FOPFlash plasmids was purchased from Upstate
Cell Signaling Solutions (Lake Placid, NY). To assay the transcriptional activity of
β-Catenin, BNL CL.2 cells were transiently transfected with a mixture (40:1) that
contained inducible (β-Catenin-LEF/TCF-sensitive reporter vector-TOPFlash) or
mutant (β-Catenin -LEF/TCF-insensitive reporter vector-FOPFlash) TCF-responsive
firefly luciferase pRLTK (Promega) vectors and siRNAs using Lipofectamine 3000
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. All
experiments were performed three times in duplicate. Firefly and Renilla luciferase
activities in the lysates were assayed with a Dual Luciferase Reporter Assay System
(Promega).
RNA pulldown assay
RNA pulldown assay was performed as described previously[ 1 ] Briefly,
biotin-labeled RNAs were transcribed in vitro with a Biotin RNA Labeling Mix
(Roche, Indianapolis, IN) and T7 RNA polymerase (Roche); they were then treated
with RNase-free DNase I (Roche) and purified with an RNeasy Mini Kit (QIAGEN,
San Diego, CA). One milligram of BNL CL.2 cell extract was then mixed with 50
pmol of biotinylated RNA. Sixty microliters of washed Streptavidin agarose beads
(Invitrogen, USA) was added to each binding reaction, which was incubated at room
temperature for one hour. The beads were washed briefly five times and boiled in
SDS buffer, and the retrieved protein was detected by a standard Western blot
technique.
Western blot analysis
Proteins from the cell lysates were prepared in a 1× sodium dodecyl sulfate buffer.
Identical
quantities
of
proteins
were
separated
by
sodium
dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose
membranes. After incubation with antibodies specific for hnRNP U(ab20666,Rabbit
IgG, Abcam), β-Catenin(8480, Rabbit IgG, Cell Signaling Technology), C-myc(5605,
Rabbit IgG, Cell Signaling Technology), Cyclin D1(2978,Rabbit IgG, Cell Signaling
Technology) C-jun(9165, Rabbit IgG,Cell Signaling Technology) or β-actin
(A2228 ,mouse IgG, Sigma-Aldrich), the blots were incubated with IRDye
800-conjugated goat anti-rabbit IgG and IRDye 700-conjugated goat anti-mouse IgG.
The proteins were detected by an Odyssey infrared scanner (Li-Cor, Lincoln, NE).
RNA Immunoprecipitation
We performed RNA immunoprecipitation (RIP) experiments using a Magna RIP
RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the
manufacturer’s instructions. The hnRNP U antibodies were used for RIP (Abcam).
The coprecipitated RNAs were detected by reverse transcription PCR and by
quantitative PCR. The primer sequences are listed in Supporting Table 1. Total RNAs
(input controls) and isotype controls were assayed simultaneously to demonstrate that
the detected signals were the result of the specific binding of RNAs to hnRNP U (3
independent experiments were performed, each in triplicate).
Immunoprecipitation
Cells were lysed in buffer that contained 50 mM Tris–HCl (pH 7.5), 150 mM
NaCl, and 1% Triton X-100 and were cleared by centrifugation. Immunoprecipitation
with an anti-hnRNP U antibody (5 μg) or an anti-β-actin antibody (5 μg) was
performed in whole-cell extracts with the Pierce Classic IP Kit (Thermo Scientific,
Rockford, IL, USA) according to the manufacturer's protocol.
Chromatin Immunoprecipitation and ChIP-sequencing
Chromatin immunoprecipitation (ChIP) was performed with an EZ ChIP
Chromatin Immunoprecipitation Kit (Millipore) according to the manufacturer’s
instructions. Briefly, BNL CL.2 cells were cross-linked with 1% formaldehyde and
then
sonicated
into
200-bp
to
1000-bp
fragments.
The
chromatin
was
immunoprecipitated with anti-hnRNP U(Abcam) and normal rabbit immunoglobulin
G (IgG). The immunoprecipitated DNA was then subjected to the generation of
sequencing libraries. The libraries were sequenced using the Illumina Hiseq2500. All
reads were aligned to the mm9 genome browser using the bowtie with default
parameters. Duplicate reads were filtered and discarded. A Model-based Analysis of
ChIP-Seq（MACS）was applied to analyze the reads relative to the enrichment regions
(i.e., peaks). The ChIP-Seq data discussed in this article have been submitted to the
National Center for Biotechnology Information (NCBI) Gene Expression Omnibus
(GEO) and are accessible through the (GEO) Series accession number GSE75335.
The detailed peak information is shown in Supporting table 2. With regards to the
validation of the ChIP-Seq data, quantitative polymerase chain reaction (PCR) was
conducted with a SYBR Green Mix (Takara Bio, Otsu, Japan). The primer sequences
are listed in Supporting Table 1.
Pharmacological activation of the Wnt/β-catenin pathway
SKL2001(Millipore) was used to activate the Wnt/β-catenin pathway. BNL CL.2
cells were incubated with 40 μM SKL2001 for 15 hours to activate the Wnt/β-catenin
pathway. DMSO(0.5%DMSO in the final cultures) was used as the negative control.
[1] Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, Shi Y, Segal E & Chang HY(2010)Long
noncoding RNA as modular scaffold of histone modification complexes. Science 329,689-693.