Univariable and multivariable analyses on LMP10 staining of TSCC and BOTSCC in relation to DFS and OS<b>.</b>

§<p>Adjusted for age and tumor stage,</p><p>*absent/low vs moderate/high,</p><p>**absent vs weak/moderate/high.</p><p>Abbreviations: HR, Hazard Ratio; CI, Confidense Interval.</p

Kaplan-Meier curves for disease free survival (DFS) and overall survival (OS) of patients with TSCC or BOTSCC, treated with intention to cure and stratified according to fraction of cells positive for nuclear expression of LMP10.

<p>A and D: DFS (A) and OS (D) for TSCC and BOTSCC irrespective of the HPV status of the tumour. B and E: DFS (B) and OS (E) of patients with HPV-positive TSCC and BOTSCC. C and F: DFS (C) and OS (F) of patients with HPV-negative TSCC and BOTSCC.</p

LMP10 expression in HPV-positive and HPV-negative TSCC and BOTSCC.

§<p>Chi-square test.</p><p>*n denotes the number of tumours.</p

Example of LMP10 nuclear and cytoplasmic expression in TSCC/BOTSCC.

<p>(A): Weak/moderate cytoplasmic expression of LMP10; (B): Moderate/high nuclear and absent cytoplasmic expression of LMP10; (C): Absent or undetectable LMP10 nuclear and cytoplasmic expression; (D): Moderate nuclear and cytoplasmic expression of LMP10. Magnification 400X.</p

Mass Spectrometry Identification of PRAME Binding Proteins.

<p>GST-PRAME binding proteins were affinity purified as described in Materials & Methods. Bands of approximately 17 kDa and 12 kDa (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058052#pone-0058052-g003" target="_blank">Fig. 3A</a>) were excised for mass spectrometry analysis. MS/MS fragmentation data were used to search the human NCBI database using the MASCOT search engine. Probability-based Mowse scores are shown.</p

Binding of PRAME to histone H3.

<p>(<b>A</b>) Co-immunoprecipitation of endogenous histones with PRAME-FLAG-6xHis isolated from extracts of transfected HEK293 cells. Input lanes (left panels) show the presence of endogenous or FLAG-tagged proteins in extracts from cells transfected with PRAME-FLAG or empty vector. After IP with anti-FLAG antibody, immunoblots were performed with the antibodies indicated. (<b>B</b>) GST-pulldown experiment showing association of histones with GST or GST-PRAME proteins. Whole cell extracts of HL60 cells were incubated with immobilised GST or GST-PRAME proteins, and bound proteins separated by SDS-PAGE. Immunoblots were performed with specific antibodies to detect association of histones H2A, H2B, H3 and H4 with GST proteins. (<b>C</b>) Direct association of histones with GST or GST-PRAME proteins. Core histone preparations were incubated with GST beads. Following extensive washing, bound histones were separated by SDS-PAGE and revealed by western blotting using specific antibodies as indicated.</p

PRAME localises to the Golgi network following LPS/IFNγ treatment.

<p>(<b>A</b>) HEK293 cells (upper panels) were transiently transfected with PRAME-EGFP (green) and stained with α-PRAME antibody (red) to confirm the identity of the overexpressed EGFP fusion protein. U2OS cells (lower panels) were cotranfected with GFP (green) and PRAME-FLAG (red). Merged images indicate the extent of coincidence of the EGFP and α-PRAME signals, and nuclear DNA is indicated (blue). The right hand panels are western blots showing detection of GFP or PRAME-EGFP proteins in whole cell extracts of transfected U2OS cells. (<b>B</b>) Immunostaining of endogenous PRAME in HL60 cells using α-PRAME antibody following treatment with PBS, LPS/IFNγ or PGN/IFNγ for 4 hrs. (<b>C</b>) Immunostaining of endogenous PRAME in U937 cells with α-PRAME following treatment with LPS/IFNγ for 0, 1 and 4 hrs. (<b>D</b>) HL60 cells treated with LPS/IFNγ for 4 hrs and immunostained with α-Golgi 58K (green) and α-PRAME (red). Merged images show the extent of colocalisation of both proteins. For immunofluorescence (A–D), nuclear DNA was stained using Hoechst 33258 and images were captured using a LSM510 confocal laser scanning microscope. (<b>E</b>) Immunostaining of endogenous PRAME in HL60 cells using α-PRAME antibody following treatment with PBS or LPS/IFNγ for 4 hrs. (<b>F</b>) Quantification (n = 60) of the percentage of cells in (E) containing PRAME cytoplasmic foci in treated cells or controls. (<b>G</b>) Immunostaining of endogenous PRAME in MCF-7 cells using α-PRAME antibody.</p

Transcriptional and translational regulation of <i>PRAME</i> by PAMPs/IFNγ.

<p>(<b>A</b>) RT-qPCR measurements of <i>PRAME</i> gene expression relative to control <i>GAPDH</i> in HL60 cells in response to treatment with different PAMPs including lipopolysaccharide (LPS), peptidoglycan (PGN), muramyl dipeptide (MDP), zymosan (ZYM) and mannan (MAN) either alone (PBS) or in combination with IFNγ. Numbers on the x-axis indicate the time in hrs post-treatment. qPCR quantifications were performed in triplicate and the data shown represents the mean of two independent experiments, with error bars indicating standard deviations. The data is presented as fold induction relative to levels obtained at 0 hr (baseline). (<b>B</b>) RT-qPCR experiment performed as in (A) showing effect of pre-treatment with actinomycin D (10 μg/ml) or PBS on induction of <i>PRAME</i> transcript in HL60 cells by LPS/IFNγ (1 hour). (<b>C</b>) Association of <i>PRAME</i> transcripts with polysomes in HL60 cells following treatment with LPS/IFNγ for 0 and 4 hrs. Following cycloheximide treatment and sucrose density centrifugation of HL60 cell lysates, gradients were fractionated with continuous monitoring at 254 nm, to generate polysome profiles (top panel). RNA extracted from polysome fractions was analysed by Northern blotting and the <i>PRAME</i> transcripts visualised by phosphoimager (middle panels) and quantified by densitometry (bottom panels). <i>β-actin</i> was used as a control probe.</p

PRAME associates with the Elongin BC complex.

<p>(<b>A</b>) SDS-PAGE and silver staining showing affinity capture of proteins from HL60 whole cell extracts (WCE) by immobilised GST or GST-PRAME proteins. GST and GST-PRAME proteins are indicated. Putative PRAME-specific bands are indicated and bands of approximately 12 kDa and 17 kDa were excised for mass spectrometry analysis. (<b>B</b>) Co-immunoprecipitation of PRAME with Elongin complex components. Whole cell extracts of HEK293 cells transfected with PRAME-FLAG-6xHis (or empty vector control) applied to anti-FLAG sepharose beads as described in Materials and Methods. After extensive washing, co-purified PRAME and E3 ubiquitin ligase complex components were detected by western blotting using specific antibodies as indicated. (<b>C</b>) GST-pulldown experiment showing binding of ELB and ELC proteins in HL60 whole cell extracts to GST or GST-PRAME proteins. The top panel is a Coomassie-stained gel showing the input whole cell extract, and the purified GST and GST-PRAME proteins. The lower panels are western blots revealing PRAME, ELB and ELC proteins bound to GST proteins. (<b>D</b>) GST-pulldown experiments revealing interactions of ³⁵ [S]-labelled <i>in vitro</i> translated human ELC (hELC), <i>C.elegans</i> ELC (wELC), <i>C.elegans</i> ELC (L47D-L49D-Y88D-Y91D) (wELC mutant) and <i>C.elegans</i> ELB proteins with GST or GST-PRAME. (<b>E</b>) Yeast two hybrid assays of LexA-PRAME interactions with GAL4 AD-fused human ELC (hELC) or <i>C.elegans</i> proteins (wELB, wELC, wELC mutant). Western blots of the HA-tagged elongin fusion proteins are also shown. Reporter activity is expressed as β-galactosidase activity normalised to amount of protein in the extracts. (<b>F</b>) Immunofluorescence staining showing subcellular localisation of endogenous ELC, ELB and CUL2 proteins in HL60 cells. (<b>G</b>) Immunofluorescence staining showing colocalisation of endogenous ELC and PRAME proteins in HL60 cells following treatment with LPS/IFNγ for 4 hours.</p