Additional file 8: Figure S7. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

Effect of cycloheximide, a translational inhibitor, on the abundance of ZFAS1 and GAS5. Relative expression of genes was measured by qPCR using total RNA extracted from each cell. 18S and 28S rRNA transcripts were used to normalise the expression of ZFAS1 and GAS5. The Y axis represents the fold change relative to time 0. The X axis shows treatment time. Error bars are SEM of three biological replicates, p values were calculated using Student’s t test. (PDF 136 kb

Additional file 9: Table S2. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

List of primers used in these experiments. The sequences are 5′ to 3′. (DOC 43 kb

Additional file 5: Figure S4. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

Effect of ZFAS1 knockdown on cell phenotype. (A) Proliferation rates of cells transfected with vectors expressing control (scrambled RNA) and ZFAS1-specific shRNA BC2 and BC3 as determined by SRB assay. Error bars are SEM, n =2. (B) Size of cells expressing control (scrambled) and ZFAS1-specific shRNA BC2 was determined using a Coulter electronic particle counter. (C) Nascent protein synthesis as quantified by uptake of the fluorescent amino acid analogue, Click-iT® AHA, in ZFAS1 knockdown BC2 and scrambled control cells, n = 2. (PDF 451 kb

Additional file 7: Figure S6. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

Effect of puromycin, a translational inhibitor, on the abundance of ZFAS1 and GAS5. Relative expression of genes was measured by qPCR using total RNA extracted from each cell. 18S and 28S rRNA transcripts were used to normalise the expression of ZFAS1 and GAS5. The Y axis represents the fold change relative to time 0. The X axis shows treatment time. Error bars are SEM of three biological replicates, p values were calculated using Student’s t test. (PDF 172 kb

Additional file 15: Figure S12. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

ZFAS1 is associated with actively translating ribosomes. (A) Polysome distribution of MDA-MB-468 cell lysates as separated on a 7–47 % sucrose gradient. Absorbance at 260 nm is shown on the Y axis. Fractions from the top of the gradient to the bottom are shown from left to right on the X axis. Fractions were collected in 36 equal volumes, of which every third was used for RNA extraction, and cDNA synthesised for PCR. (B) Polysome distribution of MDA-MB-468 cell lysate separated on a 7–47 % sucrose gradient containing EDTA instead of MgCl2. Loss of the polysome peak is observed, together with a leftward shift of the ribosome subunits. (C) and (D) Quantitative expression of ZFAS1 and GAPDH measured by qPCR relative to 18S and 28S rRNAs prepared with and without the addition of EDTA. Arrows indicate where ribosomal features are observed on profiles in relation to fraction number. (PDF 536 kb

Additional file 14: Figure S11. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

The presence of ZFAS1, 18S and 28S expression were assessed by qPCR using fractions derived from dissociated ribosomes (Fig. 4i). Red bars show the samples derived from the peak of the graph in Fig. 4Ai for 40S and 60S subunit. (PDF 235 kb

Additional file 2: Figure S2. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

Analyses of ZFAS1 in breast cancer samples derived from TCGA. A(i) Expression of ZFAS1 in normal breast (n = 113) and breast cancer (n = 1069) samples. (ii) Expression of ZFAS1 by tumour subtype based on PAM50 classification. ZFAS1 is more highly expressed in normal tissues compared to basal and HER2 breast cancer subtypes. (iii) Expression of ZFAS1 in ER+ (n = 601) and ER- (n = 179) breast cancer samples. Unpaired Student’s t-test showed that ZFAS1 was differentially expressed according to estrogen status. (B) Kaplan-Meier plot generated from http://www.oncolnc.org/ of TCGA breast cancer data set. High expressers are those 50 % of patients with the highest ZFAS1 expression, and low expressers are those 50 % of patients with the lowest ZFAS1 expression. High expressers of ZFAS1 do not show altered survival up to 6000 days. (C) Gene expression of candidate ribosomal proteins by tumour subtype based on PAM50 classification. Unpaired student’s t-test relative to normal tissue samples was used to calculate P values. (PDF 475 kb

Additional file 13: Figure S10. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

Primer efficiency of ZFAS1 and ZNFX1. A) The primer efficiency test for the primers used in the experiments. B) Slopes of standard curve indicate PCR efficiency for ZFAS1 and ZNFX1 primers sets. The X axis represent the log of dilution. Y axis shows Ct values. C) Correlation between the expression of ZFAS1 and ZNFX1 in two different primer sets in panel of cell lines (PDF 317 kb

Additional file 1: Figure S1. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

ZFAS1 is unlikely to encode a protein. Ribosome occupancy derived from multiple ribosome profiling studies according to the GWIPS database is mapped to ZFAS1. Red peaks from ribosome profile indicate the level of ribosome occupancy whereas green peaks from mRNA seq coverage indicate the level of transcription of a particular gene region. ZFAS1 is indicated in blue, with numbers indicating nucleotide number for each exon above the gene layout. Potential open reading frames, shown in pink, were predicted using ExPASy and mapped to the genomic layout of ZFAS1. Peaks corresponding to ribosome occupancy were then overlaid with ORFs, with the peaks mapping to snoRNAs in the intronic regions of ZFAS1. (PDF 271 kb

Effect of ectopic expression of <i>SOX2OT</i> on cell growth.

<p>A) The proliferation rates of cells transfected with control vector and plasmid containing the <i>SOX2OT</i> gene were measured after 1, 3, 5 and 7 days of culture. B) Proliferation rates of cells transfected with control vector or plasmid containing the <i>SOX2OT</i> gene, in monolayer and suspension culture, as well as in soft agar. * and ** represent <i>p</i> values of &lt;0.05 and 0.01 respectively. C) Image of MDA-MB-231 cells grown in soft agar for two weeks. The top panel represents the colony form in cells with ectopic expression of <i>SOX2OT</i>. The bottom panel represents cells containing the control vector. D) Effect of ectopic expression of <i>SOX2OT</i> on cell cycle distribution. The cells were treated with and without paclitaxel for 24 h and stained with PI. Following FACS analysis, DNA histograms were analyzed using ModFit LT. The data are representative examples for duplicate tests.</p