The putatively encoded protein isoform smLRP1 is expressed in human tumour cell lines.

<p>Tumour cell lysates (40 μg) and purified LRP1 (1 μg) were subjected to SDS-PAGE under non-reducing conditions and Western blotted using monoclonal murine anti-smLRP1 and anti-LRP1 antibodies targeting the smLRP1 C-terminus (32 kDa) and LRP1 α-chain (515 kDa), respectively. GADPH expression served as control. (<b>A</b>) In the absence of secondary anti-mouse antibody (1:5,000), no smLRP1 protein band (32 kDa) could be detected in A459. (<b>B</b>) LRP1 comprises an α- (515 kDa) and β-chain and is co-purified with RAP. (<b>C</b>) Probing purified LRP1 with anti-LRP1 detected the α-chain with a molecular weight of 515 kDa. (<b>D</b>) Contrary, probing both LRP1 and A549 lysate with anti-smLRP1 no binding to the LRP1 α-chain was observed, whereas a smLRP1 protein band (32 kDa) was detected in A549. (<b>E</b>) RNAi-mediated transient knockdown of smLRP1 mRNA expression in U87-MG for 48 h resulted in decreased smLRP1 protein expression compared to nonsense control (ns; N = 3). <b>F</b>) The smLRP1 protein (32 kDa) was detected in 12 different human tumour cell lines. <i>Abbreviations</i>: LRP1, low density lipoprotein receptor-related protein 1; RAP, receptor associated protein; ns, nonsense control.</p

Protein sequence features of smLRP1.

<p>Protein sequence features of smLRP1.</p

LRP1 and smLRP1 are differentially expressed in tumour cell lines and healthy tissues.

<p>(<b>A</b>) Quantitative Real-time PCR showed a shift in relative expression of LRP1 and (<b>B</b>) smLRP1 in tumour cell lines. (<b>C</b>) Comparing expression of smLRP1 relative to LRP1 (ratio smLRP1/LRP1) in 5 human tissues and their corresponding tumour cell lines indicated a shift in expression in favour of smLRP1 in most tumour cell lines. Results are presented as mean±SEM of 3–16 biological replicates. Significant results are highlighted accordingly: *, <i>p</i><0.05; **, <i>p</i><0.01, and ***, <i>p</i><0.001.</p

LRP1 and smLRP1 mRNA expression is altered in primary HNSCC compared to mucosa.

<p>(<b>A</b>) <i>In vitro</i> results for HNSCC cell lines were validated by screening paired mucosa and HNSCC specimen (N = 16) for smLRP1 and LRP1 showing decreased mRNA expression of LRP1 and smLRP1. Relative gene expression was referred to GAPDH and RPL27. <b>(B)</b> The ratio smLRP1/LRP1 in HNSCC was compared to the corresponding mucosa. Results are presented as mean±SEM of 14–16 biological replicates. Significant results are highlighted accordingly: *, <i>p</i><0.05; **, <i>p</i><0.01, and ***, <i>p</i><0.001.</p

Comparative sequence analysis of LRP1 and its truncated splice variant smLRP1.

<p>Comparative sequence analysis of LRP1 and its truncated splice variant smLRP1.</p

LRP1 and its truncated splice variant smLRP1 are ubiquitously expressed in human tissues and tumour cell lines.

<p>(<b>A</b>) Human tumour cell lines, (<b>B</b>) healthy tissues and PBMCs were screened for LRP1 and smLRP1 mRNA expression in PCR using GAPDH as control.</p

LRP1 and protein isoform smLRP1 are expressed in tumour cells.

<p>Tumour cells were fixated with 4% PFA and permeabilized with Triton X-100. (<b>A</b>) Co-staining of native smLRP1 using polyclonal anti-smLRP1 (rabbit) and Flag-smLRP1 after transfection with pcDNA3.1_Flag-smLRP1 vector showed concordant localisation of the native and Flag-smLRP1 protein in A549. Staining of (<b>B</b>) native LRP1 α-chain and over-expressed Flag-smLRP1 targeting the N-terminal Flag-tag in A549 and U87-MG showed localisation in vesicles in the cytoplasm along the plasma membrane.</p

Characterisation of HNSCC patients.

<p>Characterisation of HNSCC patients.</p

The Low density lipoprotein receptor-related protein 1 and its truncated isoform “smLRP1”.

<p>(<b>A</b>) The human gene <i>LRP1</i> is located on chromosome 12q13-14. Alternative splicing produces the alternative transcript BC072015.1 (NCBI database), called smLRP1, a truncated mRNA comprising 6 protein-coding exons identical to LRP1 and an alternative exon 7 (exon 7a) containing a stop codon and a polyadenylation signal evading nonsense-mediated decay. (<b>B</b>) The LRP1 comprises an extracellular α-chain (515 kDa) containing 4 ligand binding clusters and a non-covalently associated transmembrane-spanning β-chain (85 kDa), whereas a molecular mass of 32.4 kDa was calculated for smLRP1. <i>Abbreviations</i>: LRP1, lipoprotein receptor-related protein 1; CR, complement like, cysteine-rich sequence repeats; EGF-like, epidermal growth factor-like.</p

LRP1 and the putative protein isoform smLRP1 share high sequence identity.

<p>Sequence alignment of LRP1 (NP_002323.2) and smLRP1 (AAH72015.1) was done using online tool <i>Clustal Omega</i> (<a href="http://www.ebi.ac.uk/Tools/msa/clustalo/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalo/</a>).</p