B₁R WT is more stable than B₁R SV under basal conditions.

<p>Actinomycin D (Act D) mRNA decay of B₁R WT and SV transcripts in H2126 measured at 0, 1, 3, 5h using real time PCR (Act D treatment at concentration of 5 µg/mL). Data plotted is mean±SEM from four independent experiments each performed at least in duplicates. Half-life of mRNA can be roughly estimated by determining the time required to reach 50% transcript level (shown by dotted lines). For more accurate assessment, the trendline equations obtained by plotting the graph are used to determine the half-life. In this graph, the equation for B₁R WT is y = 100e^−0.213x while for B₁R SV is y = 100e^−0.344x, where y is set to 50 (indicating 50% of transcript remaining) which will allow the calculation of x (indicating time required to reach 50% transcript level). From these equations, the half-life of B₁R WT is 3.28 hr and 2.02 hr for B₁R SV.</p

B₁R SV does not affect translation efficiency.

<p>Translation efficiency of B₁R WT and SV 5′ UTR measured using luciferase expression normalised to Renilla expression over time. Transfection of WT-luciferase and SV-luciferase constructs into normal lung bronchial epithelial, 16HBE (A), and lung adenocarcinoma, H2126 (B). Results are the average of five experiments with error bars representing SEM. There was no significant difference between the translation efficiency of B₁R WT and B₁R SV.</p

B₁R transcript is differentially expressed across a range of cell lines.

<p>B₁R mRNA expression was normalised to housekeeping gene SOD1 in human pulmonary cell lines as quantified by real time PCR. Data represents mean ± SEM from 3 independent experiments, each performed in duplicate.</p

Deletion B₁R promoter constructs in HIGH expressing H2126 and LOW expressing 16HBE reveal regulatory regions.

<p>Summary of results from deletion constructs of B₁R regulatory regions (A). The size of each regulatory region is indicated relative to start of exon III of B₁R (+1). PRE = positive regulatory element, NRE =  negative regulatory element. Relative luciferase activity of promoter deletion constructs transfected into human lung adenocarcinoma H2126 (B) and human bronchial epithelium 16HBE (C). Data presented as mean with error bars representing SEM. Data was analysed using one-way ANOVA and Tukey's post-hoc test on 4 independent experiments, each performed using at least triplicates. *p≤0.05 was considered statistically significant. Activity of CP-E2I2-Luc construct containing the B₁R 5′ promoter and −1020 bp to +1 was set to 1.</p

B₁R WT and SV expression following DAKD stimulation.

<p>Quantitative real-time PCR measurements of time (0, 3, 6 and 24 hr) and dose effect of DAKD (100 nM and 1000 nM) on B₁R WT (A) and SV (B) expression in H2126 and on B₁R WT expression in 16HBE (C). B₁R mRNA level at 0 hr was set to 1. Data from 4 experiments performed in duplicates with mean ± SEM represented. DAKD treated samples were compared with non-treated, media only (NT) for each time point. Data was analysed using unpaired Student's t-test where *p<0.05 is considered statistically significant. **p<0.001</p

5′RACE PCR analysis of H2126 cDNA reveals multiple products.

<p>H2126 cDNA was amplified using the GeneRacer 5′nested primer and RT Rev 2 primer (A). Expected product size was 450 bp although at least 5 other bands were observed. Lanes 1 and 2: H2126 cDNA, Lanes 3 and 4: no template control. <b>Major transcription start sites (TSS) identified</b> in this study are labelled relative to translation start site (ATG) of NCBI published sequences of B₁R (B). TSS of transcript D identified in this study is located 12 bp upstream of TSS on NCBI (NM_000710) but matches TSS identified by Yang & Polgar (1996)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087175#pone.0087175-Yang3" target="_blank">[23]</a>. In addition to the full-length wild type B₁R transcript, a splice variant of B₁R (transcript D and E) was also identified in this study. The TSS of this splice variant was at two primary locations; 12 bp and 4 bp upstream of NCBI sequence. Schematic presentation of identified wild type (WT) and splice variant (SV) transcripts and position of primers used in RT-qPCR to specifically amplify WT (B1R WT F) and SV (B1R SV F) (C). Forward primers are spanning the splice sites while common reverse primer (B1R Rev qPCR) located in exon 3 was used for amplification of both transcripts.</p

B₁R splice variant (B₁R SV) transcript and heteroduplex band is present in several cell types.

<p>Amplification of B₁R WT and SV using primer set RT Fow and RT Rev (A) and RT Fow2A and RT Rev 1 (B). Amplification of loading control housekeeping gene superoxide dismutase 1 (SOD1) (C). Amplification of B₁R WT and SV from human leucocytes using primers RT Fow and RT Rev (D) and amplification of SOD1 (E). A representative image of additional heteroduplex band located between the WT and SV band in HFLF. Three extra PCR cycles were used to match conditions used in G, but without addition of 10X fresh PCR mix (F). Heteroduplex band removed after addition of 10X fresh PCR mix followed by 3 PCR cycles (G). Lane 1: 100 bp ladder. PCR no template control (NTC). Human leucocytes (Leuc).</p

Measures of diagnostic accuracy of pleural fluid Hsp72.

<p>Values in parentheses are 95% confidence intervals.</p><p>LR, likelihood ratio; AUC, area under ROC curve.</p>*<p>This figure represents three times the upper normal limit for serum LDH.</p

Hsp72 levels in peritoneal lavage are elevated following intraperitoneal injection of mice with <i>Streptococcus pneumoniae</i>.

<p>BALB/c mice were given a single intraperitoneal injection of live <i>Streptococcus pneumoniae</i> D39 strain (∼1×107 CFU in 0.1 ml of saline; n = 11) or saline as a control (n = 10). The peritoneal cavity was lavaged 7 (n = 6) and 17 hr (n = 5) post-injection with 1 ml PBS for quantification of Hsp72 protein. The results are presented as the fold-increase in Hsp72 levels over the mean Hsp72 level in mice injected with saline alone. An approximately 2- and 2.5-fold increase in Hsp72 was shown peritoneal lavage following infection with <i>S. pneumoniae</i> for 7 and 17 hr, respectively (p<0.01 for both).</p