PSA and Gleason sum scores of SCT population.

<p>(a) Dot blot showing the pre and post-SCT nadir PSA for the entire patient cohort, (b) Bar chart representing the numbers of patients with different Gleason sum scores prior to SCT.</p

Simplified analysis of gland length and the largest iceball dimension against IPSS scores.

<p>Significant association between gland length and iceball length with (mean and maximal) IPSS scores were observed. (1_C and 2_C denotes 1^st and 2^nd cycles, respectively; n  =  patient numbers).</p

Disease free survival by (a) Pre-SCT PSA, (b) Gleason Score and (c) PSA nadir.

<p>Disease free survival by (a) Pre-SCT PSA, (b) Gleason Score and (c) PSA nadir.</p

Summary of multivariate analysis.

<p>Multivariate analysis of SCT parameters reveals highly significant association between dimensions of gland length and the iceball with patients' IPSS scores. (1_C and 2_C denotes 1^st and 2^nd cycles, respectively; Right and left lobe signified the nadir temperature recorded below 0°C; Iceball Length signifies the dimension of iceball parallel to the anterior rectal wall, measured in mm, at the end of each freeze cycle; n  =  patient numbers).</p

Association between gland length and maximal length of iceball recorded with patient IPSS outcome.

<p>(Max Lobe represents the lowest temperature below 0°C recorded in the prostate; Max Iceball is taken as the longest dimension of iceball recorded for the entire procedure; n  =  patient numbers.).</p

Post-SCT complications and the number of patients affected by each complication.

<p>Post-SCT complications and the number of patients affected by each complication.</p

Summary demographics of our series of patients who received SCT.

<p>Breakdown of patient demographics and tumour parameters from the three cryotherapy centres can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069243#pone.0069243.s004" target="_blank">Table S1</a>. Please note that not all categories add up to 283, since data was incomplete for some patients as regards Pre Rx Gleason, Pre Rx PSA and Clinical T score. (Rx  =  cryotherapy).</p

Validation of AR detection with flow cytometry.

<p><b>A)</b> Percentage of cells staining above a no antibody control for either isotype antibody (hollow points) or PG-21 AR antibody (solid points) in LNCaP (circles) or PC3 (triangles) across a dilution series. <b>B)</b> Representative staining patterns for PC3 (upper dotplots) and LNCaP (lower dotplots) for either 1∶200 isotype antibody (left dotplots) or 1∶200 PG-21 AR antibody (right dotplots) of equivalent concentrations. Gates set according to isotype control. <b>C)</b> Left dotplot representative of staining of LNCaP with isotype control, right dotplots representative of AR staining in non-transfected LNCaP (upper), LNCaP transfected with scrambled siRNA (middle dotplot) and LNCaP transfected with AR siRNA (lower). Gates were set according to an appropriate isotype control. <b>D)</b> Percentage of cells staining positive for AR relative to an isotype control in non-transfected LNCaP, LNCaP transfected with scrambled siRNA and LNCaP transfected with AR siRNA. Error bars represent standard error of the mean for n = 3. <b>E)</b> Western blots of AR expression for non-transfected LNCaP, LNCaP transfected with scrambled siRNA and LNCaP transfected with AR siRNA are shown using a different AR antibody (G122-434, BD Pharmingen).</p

Strategy of enrichment for required cell types.

<p><b>A)</b> Schematic work flow for enrichment of epithelial cells for assessment of AR expression. <b>B)</b> Purity of selection by expression of the lineage specific markers CD24 (epithelial), CD146 (endothelial) and CD45 (haematopoietic) normalised to GAPDH following real-time PCR for unsorted prostate epithelia and EpCAM/HEA sorted epithelia, error bars represent standard error of the mean for n = 3. <b>C)</b> CD133/1 Sorted samples were assessed for purity by co-expression of the CD133/2 epitope, confirming that these two epitopes are co-expressed in the prostate and that our CD133 selection efficiently enriches for CD133^+VE cells: Upper dotplot representative of CD133/2 staining for unsorted α₂β₁^HI epithelial cells; lower left dotplot representative of CD133/2 staining for α₂β₁^HI CD133/1^–VE cells; lower right dotplot representative of CD133/2 staining for α₂β₁^HI CD133/1^+VE cells. Gates are set according to appropriate isotype controls. <b>D)</b> Confirmation of CD133 enrichment with real-time PCR. CD133 expression is shown normalised to GAPDH, error bars represent standard error of the mean n = 10.</p

AR expression and activity within the prostate epithelial hierarchy of differentiation.

<p>A) Representative images of expression of CD133 and AR counterstained with DRAQ5™ within prostate EpCAM^+VE α₂β₁^HI CD133^+VE (Left panel), α₂β₁^HI CD133^–VE (central panel) and α₂β₁^LOW CD133^–VE cells (right panel). <b>B)</b> Expression of the AR regulated genes PSA, KLK2 and TMPRSS2 normalised to GAPDH (n = 10) (p<0.001 comparing α₂β₁^HI and α₂β₁^LOW). Error bars represent standard error of the mean.</p