SUPPLEMENTAL FIGURE LEGEND
Supplemental Figure S1. NOL7 is undetectable in cytoplasmic fractions. Cell lysate from SiHa cells expressing V5-tagged wild-type NOL7 were separated into nuclear and cytoplasmic fractions and subjected to western blotting. As a loading and fractionation control, nuclear SP1 and cytoplasmic
GAPDH expression were also tested. NOL7 was not found at any detectable level in the cytoplasm.
Supplemental Figure S2. NOL7 or HuR expression does not regulate endogenous levels of either gene. A) Parental 293T and SiHa cells, as well as SiHa cell stably expressing GFP, NOL7, or HuR were analyzed by real-time PCR for endogenous mRNA levels. Data are represented as mean ± SEM of three independent assays from two individual clones for each cell line. B) SiHa stable cell lines were assessed for V5-fusion protein expression as well as for endogenous HuR expression. β-actin was included as a loading control.
SUPPLEMENTAL MATERIALS AND METHODS
In vivo Tumor Studies.
Cells were injected into the subcutaneous tissue of 6-8-week-old female nu/nu mice
(Charles River Laboratories, Wilmington, Maine) at a final concentration of 10 7 cells in 0.1ml of PBS
(n=6 per group). Tumor growth was monitored with a caliper.

Tissue Culture
HEK293T cells were obtained from the ATCC (Manassas VA) and grown in DMEM supplemented with 10% fetal bovine serum (FBS), 100 µg/ml penicillin/streptomycin and 200
µg/ml L-glutamine. For all transfection experiments, cells were transfected with Lipofectamine
2000 according the manufacturer’s instructions when cells were approximately 90% confluent
(Invitrogen, Carlsbad CA). Twenty-four hours after transfection, cells were split to ensure approximately 50% confluency for assays. For stable cell lines, cells were selected in 1.2 µg/ml puromycin (Sigma-Aldrich, St. Louis MO) for three weeks and stable pools were ring cloned to obtain individual stable cell lines.
SiHa cells were obtained from the ATCC (Manassas VA) and grown in RPMI supplemented with
10% FBS and 100 µg/ml penicillin/streptomycin. Cells were transfected with Xfect (Clontech,
Mountain View CA) according the manufacturer’s instructions when cells were approximately 70% confluent. For stable cell lines, cells were selected in either 2.5 µg/ml puromycin (Sigma-Aldrich, St.
Louis, MO) or 20 µg/ml blasticidin (Invitrogen, Carlsbad CA) and stable pools were ring cloned to obtain individual stable cell lines.
Cloning and Expression Constructs
Expression Constructs. GFP wild-type and mutant constructs were cloned as previously described
(Zhou et al 2010). GFP, NOL7, and HuR were cloned with tandem C-terminal V5-His
6
tags using the
Gateway cloning system from Invitrogen (Carlsbad, CA). All TOPO and LR cloning reactions were performed as described by the manufacturer. The coding sequence for GFP, NOL7, and HuR were
PCR amplified using Platinum Pfx polymerase (Invitrogen, Carlsbad CA) using the following primers: GFP, 5’-CACCATGGCTAGCAAAGGAGAAGAAC-3’ and 5’-TTTGTAGAGCTCATCCATGCC-3’;
NOL7, 5’-CACCATGGTGCAGCTCCGACC-3’ and 5’-CTTCTTAGTTTTCATCTTTCTGACCATCCACCG-3';
HuR, 5’-CACCATGTCTAATGGTTATGAAGACCAC-3’ and 5’-TTTGTGGGACTTGTTGGTTTTGAAGG. PCR

products were then TOPO cloned into pENTR-SD-D-TOPO. The pENTR constructs were transferred to the pEF-DEST51 vector.
In Vitro Biotinylated Transcript Templates. The 3’UTR of TSP-1 was PCR amplified from cDNA using the following primers: 5’-TCATCAAATTGTTGATTGAAAGACTG-3’ and 5’-
AAATTTCCATATGATTTATTGTTGTTC-3’. The random, nongenic construct was PCR amplified from the pSGG-R01 vector (SwitchGear Genomics, Menlo Park CA) using the following primers: 5’-
AGCTCTTTGGAACATGACGGA-3’ and 5’-CCAGCCAAACACAGGTGGTTTACA-3’. PCR was performed with Platinum Taq polymerase and TA cloned into the pCRII vector. The sense clone was identified by sequencing.
IP-Northern Probe Template. The template for the in vitro transcription of the TSP-1 mRNA probe was generated by PCR amplification of a 240-bp region of the TSP-1 3’UTR using the following primers: 5’-CAGAGGCCAAAGCACTAAGG-3’ and 5’TGAGTAAGGGTGGGGATCAG-3’. PCR was performed with Platinum Taq polymerase and TA cloned into the pCRII vector. The antisense clone was identified by sequencing.
Fluorescence Microscopy
Parental SiHa cells or SiHa cells expressing vector control, wild-type NOL7, or localization mutants were seeded on 4-well chamber slides. After 36 h, cells were fixed with 4% paraformaldehyde for 30 min, rinsed with PBS, and mounted in DAPI-containing media (Vector
Labs, Burlingame CA) according to the manufacturer's instructions. Fluorescence images were obtained using a Zeiss Axioplan microscope and Openlab capturing software (Improvision,
Lexington, MA, USA).

Quantitative Real-Time PCR
RNA levels were measured by real-time quantitative PCR using the Ag-Path One Step RT-PCR kit
(Ambion/Applied Biosystems, Austin, TX). For each, 30ng total RNA was amplified on the CFX-1000
(Bio-Rad Laboratories, Hercules, CA) at 45°C for 10 min, 95°C for 10 min, followed by 40 cycles of
95°C for 15s and 60°C for 45s and detected using TaqMan probes against target transcripts
(Applied Biosystems, Austin, TX). TaqMan probes used include GAPDH, 18S, firefly luciferase, NOL7,
HuR, and 18S rRNA. Relative expression levels were calculated using the ΔΔCt method relative to
18S. Statistical differences were calculated as indicated.
Analysis of Cell Line Expression
Total RNA from parental 293T and SiHa cells as well as stable SiHa cell lines expressing GFP,
NOL7, or HuR were collected by Trizol extraction according to manufacter instructions. Transcript expression was measured by real-time quantitative PCR using the Ag-Path One Step RT-PCR kit
(Ambion/Applied Biosystems, Austin, TX) and TaqMan probes against NOL7, HuR, and 18S rRNA
(Applied Biosystems, Austin, TX). Western blotting was performed on total cell lysate as described.
Mass Spectroscopy
HEK293T cells expressing vector control or NOL7-V5 were separated by sucrose gradient ultracentrifugation as described. The approximately 50-100S fractions were pooled from each and subjected to α-V5 immunoprecipitation as described (Supplemental Figure S1A). The immunoprecipitates were separated by SDS-PAGE. The gel was stained with colloidal coomassie and washed three times with sterile water (Supplemental Figure S1B). Each lane was cut into 10 gel slices and analyzed by tryptic digestion followed by chromatographic analysis using the

LTQ-FT LC/MS/MS. MS/MS samples were analyzed using Mascot and Sequest. Scaffold was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (Keller et al 2002). Subsequent protein identifications were assigned by the Protein Prophet algorithm (Nesvizhskii et al 2003). Curation of MS data was performed in sequential steps (Supplemental Figure S1C) first accepting hits if they contained at least one identified peptide that could be established at greater than 88.0% probability. This cutoff was dictated by the maximum stringency that could be applied to the dataset to detect NOL7. Any proteins that were identified in the vector control were removed from analysis. The data sets were then exported fro m the Scaffold software and manually curated.
Western Blotting
All proteins were separated on SDS-PAGE gels and transferred to ImmunoBlot PVDF membrane
(Bio-Rad, Hercules CA). Membranes were blocked in 5% milk-TBST for 2 hours at room temperature and probed overnight using the following primary antibodies: V5, Sigma-Aldrich Cat
#V8137 - 400ng/ml; NCL, Sigma-Aldrich Cat#N2662 - 2 ng/ml; NPM Epitomics Cat#20411 -
2ng/ml; SFRS5, Sigma-Aldrich, Cat#S2695 - 300ng/ml; SF2/ASF, Sigma-Aldrich, Cat#S2195 -
10ng/ml; CPSF2, Sigma-Aldrich, Cat#C1747 - 3µg/ml; XRN2, Bethyl Laboratories Cat# A301-102A -
200ng/ml; CNOT3, Bethyl Laboratories Cat#A302-156A, 20ng/ml; EXOSC10, Sigma-Aldrich
Cat#P4124, 500ng/ml; HuR, Epitomics -Cat#T3602, 400 ng/ml; SP1, Millipore Cat#17-601,
225ng/ml, GAPDH, Cell Signaling Technologiy, Cat#2118S, 25ng/ml. Goat α-rabbit HRP (Cell
Signaling Technology, Danvers MA) secondary was probed at 10ng/ml for 1 hour at room temperature. Blots were visualized using Pierce SuperSignal West Dura substrate.

Sucrose Gradient Ultracentrifugation
Twelve hours prior to centrifugation, continuous gradients were prepared in mammalian sucrose gradient buffer [50 mM Tris-HCl, pH 7.6; 80 mM KCl, 5 mM Mg(C
2
H
3
O
2
)
2
, 2% sucrose, protease inhibitors]. Gradients were poured manually and allowed to diffuse at 4°C. Immediately prior to ultracentrifugation, approximately 1x10 8 HEK293T cells at 50% confluency were collected by trypsinization and washed twice with ice cold PBS. Cells were pelleted by centrifugation and resuspended in sucrose gradient buffer. Cell pellets were sonicated 6x30s at 30% power. Lysates were then cleared by centrifugation. Protein concentration was determined by Bradford assay.
RNase treatment was performed on lysates prior to loading by incubation with 500µg/ml RNase A and 50µg/ml EDTA at 37°C for 1 hour. For gradient-western experiments, 500µg total protein was separated on a 10-30% gradient for 8 hours, while for gradient-MS experiments 2 mg total protein was separated on a 5-25% gradient for 4 hours. All gradients were separated at 27,500 rpm at 4°C on Beckman LM-80 Centrifuge. Equivalent fractions were collected manually with capillary tubes and 200 µl per fraction was transferred to a 96-well plate and measured for absorbance at 260 nm.
Immunoprecipitation
SiHa cells stably expressing GFP-V5 or NOL7-V5 were resuspended in sucrose lysis buffer and lysed by freeze-thaw. Lysate was cleared at 10,000xg for 10 min and lysate concentration was determined by Bradford assay. Lysate was diluted to 1µg/µl and 250 µg total protein was mocktreated or digested with 250ng/µl RNase A at 37C for 30 minutes. After digestion, lysates were chilled on ice and then incubated overnight at 4°C with 25 µl α-V5 agarose beads (Sigma-Aldrich, St.
Louis MO). Beads were pelleted at 1,000xg for 1 min and washed thoroughly in lysis buffer.
Immunoprecipitated proteins were eluted from the beads by boiling in SDS sample buffer. Total
RNA was extracted from digested lysates, reverse transcribed using the Superscript III One-Step

RT-PCR kit according to manufacturer’s instructions. Purified cDNA was amplified with 18S specific primers and visualized by ethidium bromide staining.