Supplementary Material
Materials and Methods
Yeast Two-Hybrid
The Matchmaker Gold Yeast Two-Hybrid System (Clontech) was used to screen a HeLa cell universal human cDNA library. 327 positives were initially selected under the lowest stringency. A highest stringency selection resulted in the isolation of 18 D1 isolates and 23 D2 isolates. Proteins listed in the table in
Figure 1A were re-confirmed as positive for interacting to the respective CIP2A domain by plasmid isolation and re-confirmation under the highest stringency. To confirm the CIP2A-LRRC59 interaction, LRRC59 was cloned into the multiple cloning site (MCS) of the pGBKT7 vector (Clontech, 630489) containing the
GAL4-DNA Binding Domain and N-terminal Myc tag and CIP2A was cloned into the MCS of the pGAD T7 vector (Clontech, 630442) containing the GAL4-
Activation Domain and an N-terminal HA tag. Both vectors were autonomously replicated in DH10b E. coli and transformed into the Matchmaker Gold Yeast
Two-Hybrid Y2H strain (Clontech).
Cell Culture, Cloning, and Transfection
The human prostate carcinoma cell line PC-3 (CRL-1425) and DU145 (HTB-
81) were grown in Ham’s F12K Kaighn’s medium (Invitrogen, 21127-022) and
Minimum Essential Medium Eagle (Sigma, M5650) supplemented with 10% fetal calf serum (Invitrogen, 16140071). RWPE-1 (CRL-11609), human prostate immortalized cells were grown in Keratinocyte-SFM medium supplemented with
10% fetal calf serum and growth supplements. All cells were grown at 37ºC in a
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5% CO
2
incubator. All cell lines were purchased from ATCC and were authenticated in November or December of 2013 via STR analysis conducted at
Genetica DNA Laboratories, Inc (www.genetica.com). For siRNA experiments,
PC-3 cells were seeded in 6-well plates at a density of 4-5x10 5 cells/well and transiently transfected with 100 nM of each of the targeted siRNA (Dharmacon, see Supplemental Table S1) or in combination utilizing Lipofectamine RNAiMax
(Invitrogen, 13778-075). RNA and protein were isolated at 48 h after transfection for analysis of target gene expression.
LRRC59-cFlag was cloned by amplifying cDNA from PC-3 cells and cloned into the C-terminal Flag vector from StrataClone Mammalian Expression Vector
Systems (240230). GFP-CIP2A was cloned by amplifying CIP2A from cDNA obtained from Open Biosystems (MHS6278-211688186) and then cloning it into a pEGFP-N1 vector obtained from Clonetech.
For transient transfections, PC-3 cells were seeded in 10 cm plates 24h before transfection. Cells were transfected using Lipofectamine 2000 with 120 nM siRNA and 4
µg of plasmid DNA.
Cell Synchronization and Immunofluorescence Microscopy
Cells were treated without serum for 16 hr for G0 arrest. A double thymidine block was performed to arrest cells at G1/S phase. Cells were treated for 16 hr with 1 mM thymidine, washed, released for 8 hr, and treated with thymidine again for 16 hr. To arrest cells at G2/M phase cells were treated with 1 mM thymidine and 100 ng/mL nocodazole. The schematic of treatment is provided in
Supplemental Figure S1. PC-3 cells were washed once in PBS and fixed in 4%
2

paraformaldehyde for 15 min. Cells were washed twice in PBS and permeabilized, blocked with PBS containing 0.3% Triton-X100/10% goat serum for 1h. Primary antibodies for anti-CIP2A (Santa Cruz, sc-80659), anti-LRRC59
(Novus Biologicals, NBP1-93952), and were incubated in antibody dilution buffer
(PBS containing 1% BSA, 0.3% Triton X-100) for 1-2 h. The cells were washed three times in PBS and incubated with fluorescent secondary antibodies antimouse or anti-rabbit alexa fluor 488 (Cell signaling, 4408 or 4412 ) and antirabbit Cy3 (Jackson ImmunoResearch, 711-165-152) in antibody dilution buffer or in PBS containing 2% normal donkey serum, 0.3% Triton X-100 for 1 h. The cells were again washed three times with PBS and stained with DAPI (Cell
Signaling, 4083) for 5 min and sealed with Prolong Gold antifade reagent
(Invitrogen, P36930) and viewed under a 100x objective on a Leica DMI 6000B microscope (Leica, Germany).
To confirm cell cycle arrest, PC-3 cells were collected and washed once in
PBS and fixed in 70% ethanol overnight at 20ºC. Following fixation, cells were spun down and stained with propidium iodide (10µg/mL, Invitrogen, P3566) and
RNase A for 1h at 37ºC. Data was collected using the BD FACS Calibur
Flow Cytometer (BD Biosciences) and analyzed with FLOWJO.
Cell Proliferation Assays
For foci formation assays, PC-3 cells were counted and 20,000 cells were plated in 6-well plates. Cells were treated with 100 nM of the appropriate siRNA and Lipofectamine RNAiMax every three days for a total of 10 days. Resultant foci were stained with crystal violet (Sigma, HT901) and washed with PBS prior
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to addition of 1% SDS in ethanol. After dissolving, an OD was taken at 570 nm to quantify relative cell proliferation. Data is representative of three individual experiments and is shown as percent of control.
For bromodeoxyuridine (BrDU) assays, PC-3 cells were seeded in a 96-well plate at a density of 5x10 4 cells per well and were transiently transfected with 100 nM of each of the targeted siRNAs (Dharmacon) using Lipofectamine RNAiMax
(Invitrogen, 13778-075). After 48 hr, cell proliferation rates were determined using the ELISA BrDU kit (Roche, 11647229001) as recommended by the manufacturer. Error bars represent standard error of the mean and significance for both assays was determined using the standard student’s t test, where * p <
0.05 and *** p < 0.001.
Immublotting, Immunoprecipitation, and Immunodepletion
Protein extraction and immunoblotting for western analyses were performed as described by Pallai et al.
(2012). To complete subcellular fractionations, the
Nuclear complex co-ip kit by Active Motif was utilized using PC-3 cells.
For western analyses, the following primary antibodies were used:
α-CIP2A
(GenScript), α-LRRC59 (Novus Biologicals, NBP1-93952), α-PR65/A (Santa
Cruz, sc-61121),
α-Lamin B (Santa Cruz, ab16048), α-Flag M2-Peroxidase
(A8592, Sigma), and α-GAPDH (Abcam, ab9385) were utilized. Secondary antibodies were sheep-anti rabbit IgG-HRP (GE Healthcare, NA934V), sheepanti mouse IgG-HRP (GE Healthcare, NA931V), or donkey anti-goat IgG-HRP
(Santa Cruz, sc-2020).
Detection of signal was performed by adding
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chemiluminsecent substrate (Millopre, WBKLS0500) and visualized by exposure to a charge-couple device camera (GE ImageQuant LAS 3000).
For co-immunoprecipitation studies from cell lines, PC-3 cells were harvested 48 h post transfection and lysed using RIPA buffer (20 mM Tris pH
7.5, 150 mM NaCl, 1% NP-40, 0.5% Sodium dexycholate, 1mM EDTA, 0.1%
SDS). The lysates were sonicated (Bioruptor, Diagneode) for 10 minutes at high on a 30 s on/off cycle and then centrifuged at 13,000 rpm for 10 minutes. 150-
300
µg of protein extract was pre-cleared with a 50% Protein G and 50% Protein
A agarose beads (Millipore, 16-201, 16-157) for 2h at 4 0 C prior to incubation with one of the following antibodies at 5 – 10 µg at 4 0 C overnight: α-GFP antibody
(ab290, abcam), Normal mouse IgG (12-371, Millipore),
α-CIP2A (ab61863,
Abcam), α-LRRC59 (ab127912, Abcam). The immunocomplexes were collected with the Protein A/G agarose bead slurry, washed 3 times with wash buffer (0.1%
Triton-X in 1X TBS), and then boiled with 2X sample buffer for 10 minutes. Inputs were prepared using 15-30ug of protein (10% of IP). Samples were then subjected to western blot analysis.
For immunoprecipitation from yeast strains, proteins were extracted from yeast as follows: strains were grown to exponential log phase at 30°C in selective medium. Cells were disrupted with cold glass beads and lysis buffer (Tris base, pH 7.9 containing (NH
4
)
2
SO
4
, MgSO
4
, Glycerol, and EDTA). Lysed cells were centrifuged at 3,300 rpm for 5 min to obtain a cell-free protein extract.
300 µg of total protein extract was pre-cleared with a 50-50 mix of
Agarose A and G beads overnight at 4 0 C. The tubes were spun at 2000 x g for 2
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min and the supernatant was then incubated with 5 µg anti-HA or IgG overnight at 4 0 C. Immunocomplexes were collected with a 50-50 mix of Agarose A and G beads for 2 hr at 4 0 C. Beads were washed 3 times and proteins were eluted in
2X loading dye. Samples were then subjected to western blot analysis.
For immunodepletion experiments, PC-3 cells were co-transfected using
10 µl of Lipofectamine 2000 with 2 µg of CIP2A-GFP and 2 µg of LRRC59-Flag.
PCMV-Flag and pUB-GFP empty vectors were used as vector controls. The cells were harvested 48 h post-transfection and then 10
0 µg of protein was precleared using a 50-50 mix of Agarose A and G beads for 2 h at 4 0 C. The tubes were spun at 2000 x g for 2 min and the supernatant was then incubated with 1
µg of PPP2CA antibody (Abcam, ab33537) and 1 µg of PR65/A antibody
(Abcam, ab24728) overnight at 4 0 C. The tubes were again spun at 2000 x g for 2 min and the supernatant was incubated with 1 µg of GFP antibody overnight at
4 0 C. A 50-50 mix of Agarose A and G beads were added and incubated for 2 h at
4 0 C. The lysate was then washed with wash buffer (0.1% Triton-X in 1X TBS), then boiled with 2X sample buffer for 10 min, and samples were analyzed by western analysis as above. Antibodies for western analysis were α-PR65/A
(Santa Cruz, SC-
6115) and α-PPP2CA (Abcam, ab33537). qRT-PCR
Extraction of RNA and qRT-PCR were performed with modifications from
Pallai et al.
(2012). Five hundred nanograms of RNA were utilized for cDNA synthesis using Superscript VILO cDNA synthesis kit (Invitrogen, 11754050). The qScript SYBR green PCR kit (Quanta Bioscience, 95089-200) was utilized and
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PCR was performed according to the manufacturer's instructions using the
Stratagene 3000 real time RT cycler (Agilent Technologies). Primers utilized for
RT-PCR are described in Supplementary Table S2.
Statistical Analysis
Statistical analysis was completed using GraphPad Prism version 5.02 for
Windows, GraphPad Software, La Jolla California USA, www.graphpad.com. The data was analyzed with Oneway ANOVA or the student’s standard t test; standard errors of the mean (SEM) were calculated for all sample batches. The p values are represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.
Hour: 0 24 40 42 48 64
Seed cells
+siRNA
+ Thymidine Wash,
Release
+Noc.
+siRNA
Fix,
Image
Supplemental Figure S1.
Treatment schematic for arresting PC-3 cells at
G2/M.
Supplemental S2. Co-immunoprecipitation of endogenous proteins.
Nuclear extracts from PC-3 cells were collected and incubated with antibodies for specific proteins, as described above, and were immunoprecipitated. Samples
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were resolved by SDS-PAGE and analyzed by western analysis. Lamin B serves as a nuclear marker.
Supplemental Table S1.
siRNA sequences used for studies.
Target mRNA
CIP2A
LRRC59-1
LRRC59-2
PPP2CA
Sequence
ACAGAAACUCACGACUA
GUAAUAAACUGACUACUCU
UGAAUGAGGUCCCGGUGAA
UAACCAAGCUGCAAUCAUG
Supplemental Table S2.
Primers used for RT-PCR analysis.
Target mRNA
CIP2A
LRRC59
GAPDH
Sequence
Forward 5’-TGCCTGCTTGAAGTCCTTG-3
Reverse 5’-TAGTCGTGTGAGTTTCTGTCC-3’
Forward 5’-TAATAAACTGACTACTCTAC-3’
Reverse 5’-CCAAAGTCTGCTGGCAGCTGCT-3’
Forward 5’-TGGGCTACACTGAGCACCAG-3’
Reverse 5’-GGGTGTCGCTGTTGAAGTCA-3’
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