Target gene expression following <i>PAX3</i> silencing.

<p>Remaining expression levels (%) of PAX3 downstream target genes in melanocytes (NHEM-n and NHEM-a (I)) and melanoma cells (WM115 and M14) assessed two day post-transfection with <i>PAX3</i> siRNA. Cells were treated with 10 μM siPAX3#1 (s224172, Ambion) alone, with consistent results observed across several independent experiments. Expression levels of downstream target genes were normalised to 18S (ΔCt) and calculated relative to the negative control siRNA (ΔΔCt). Each silencing experiment was performed with a biological duplicate.</p

PAX3/<i>PAX3</i> expression in a representative panel of melanocytes and melanoma cell lines.

<p>A) PAX3 protein levels relative to β-actin. Each sample was run in duplicate on separate gels and the density of each band was assessed by densitometric scanning using the GS-800 Calibrated Densitometer (Bio-Rad). The density of each PAX3 band was compared to that of β-actin for each lane on the same membrane and the fold change calculated and graphed. The error bars represent the standard deviation between duplicate samples. B) <i>PAX3</i> mRNA expression in neonatal and adult normal human epidermal melanocytes and primary and metastatic melanoma cell lines. RT-qPCR was performed using RNA from neonatal (NHEM-n) and adult (NHEM-a (P) and NHEM-a (I)) human epidermal melanocytes as well as six primary (MM200, MM229, MM329, MM540, MM622 and WM115) and five metastatic (A2058, M14, SKMEL2, SKMEL5 and UACC62) melanoma cell lines. The level of <i>PAX3</i> expression was calculated as fold-change relative to <i>GAPDH</i> expression levels. All PCRs were performed in triplicate and average values were used to calculate fold change over <i>GAPDH</i> for each sample. All samples were run in biological duplicates and the error bars represent the standard deviation of the biological replicates. C) Immunocytochemistry of neonatal and adult human epidermal melanocytes, five metastatic melanoma cell lines and six primary melanoma cell lines. Nuclear PAX3 protein expression (red) is observed in all melanoma cell lines and primary melanocytes. Cell nuclei are stained with DAPI (blue). The scale measures 100 μM. A negative control was included with each experimental run and proved negative in each instance.</p

Neutrophil recruitment and apoptosis.

<p>(A) Neutrophil recruitment to treatments sites (left bar chart) and to within ≈200 μm of tumour (right bar chart). Treatment sites; day 2 post initiation of ingenol mebutate treatment, treatment sites were excised and processed for immunohistochemistry and stained with the neutrophil marker, anti-Ly6G. Slides were scanned and analysed by Aperio Pixel count software for brown staining (default settings) excluding areas containing tumour (as melanosomes provide a false positive signal). Two sections per mouse, 6 mice per group. Statistics by Kolmogorov-Smirnov tests (differences in variance between groups was >4). Within ≈200 μm of tumour; in sections where the tumour mass could be readily identified (by the presence of black melanosomes), brown staining surrounding the tumour (within ≈200 μm) was quantitated as above. One section per mouse, 4–5 mice per group. Statistics by Mann Whitney U test (non-parametric data distribution and differences in variance <4). (B) Images illustrating the reduced density of anti-Ly6G staining neutrophils (brown stain) within ≈200 μm of the tumour mass in anakinra versus PBS treated mice. The tumours are delineated by white lines and identified by the presence of black melanosomes. Sections are oriented with the skin (not shown) at the top, with the tumours located in the dermis. (C) ApoTag staining of the sections described in A. Six mice per group, 2/3 sections per mouse, statistics by 2 way ANOVA (parametric data distribution and differences in variance <4, drug and mouse as fixed factors, 2/3 sections per mouse as dependent variables). (Examples of the staining are shown in Figure F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153975#pone.0153975.s001" target="_blank">S1 File</a>).</p

Relapse and survival following ingenol mebutate treatment of B16 tumours grown in MyD88^-/- and C57BL/6 mice.

<p>(A) Relapse rates following (i) ingenol mebutate treatment of B16 tumours grown in MyD88^-/- mice (n = 25), (ii) ingenol mebutate treatment of B16 tumours grown in C57BL/6 mice (n = 21), (iii) placebo treatment of B16 tumours grown in MyD88^-/- mice (n = 18) and (iv) placebo treatment of B16 tumours grown in C57BL/6 mice (n = 19). Mice were scored positive when a tumour was clearly visible (≥1–2 mm in diameter). Data from two independent experiments. Ingenol mebutate treatment groups were significantly different p = 0.021, log-rank (Mantel-Cox) test. (B) Survival rates of the same mice described in A; mice were euthanized when tumours reached 100 mm². Ingenol mebutate treatment groups were significantly different p = 0.018, log-rank (Mantel-Cox) test.</p

IL-1α and IL-1β protein levels after ingenol mebutate treatment.

<p>(A, B) C57BL/6 and MyD88^-/- mice with B16 tumours were treated topically with ingenol mebutate day 0 and 1, treatment sites were excised and IL-1α and IL-1β levels were measured in extracts using BD BD™ Cytometric Bead Array (n = 6 mice per group and time point). Statistics by Kolmogorov-Smirnov tests (differences in variance >4). (C) Cultured adult human keratinocytes were treated with the indicated concentration of ingenol mebutate for 16 hours and the supernatants analysed by Western using an anti-IL-1α antibody. Arrow indicates the position of the 18 kDa bioactive form of IL-1α.</p

Relapse and survival following ingenol mebutate treatment of B16 tumours grown in C57BL/6 mice treated with anakinra.

<p>(A) Relapse rates after C57BL/6 mice bearing B16 tumours were treated with ingenol mebuate or placebo, and received daily injections of PBS or anakinra, days 1–7. (n = 9–12 mice per group). Mice were scored positive when a tumour was clearly visible (≥1–2 mm in diameter). Statistics compared + anakinra with + PBS in ingenol mebutate treated groups using the log-rank (Mantel-Cox) test. (B) Survival of the mice described in A; mice were euthanized when tumours reached 100 mm². Statistics as in A.</p

KLK4 is associated with poor outcome of patients.

<p><b>A.</b> Phase contrast images show the similar morphology of trypan blue stained KLK4-1 MCAs and ascites-derived MCAs from a serous EOC patient. Scale bar, 50 µm. <b>B.</b> IF confocal microscopy with an antibody against KLK4 (green), phalloidin (red) and DAPI (blue) in ascitic serous EOC MCAs from 2 patients; scale bars, 25 µm. <b>C.</b> Kaplan-Meier survival analysis shows the relationship between <i>KLK4</i> mRNA levels in tumor tissue samples and survival status of a cohort of 38 serous EOC patients. Left panel, progression free survival (PFS) time for patients with low <i>KLK4</i> (n = 25) and high <i>KLK4</i> (n = 13) levels (χ² = 8.3, <i>p</i> = 0.004). Right panel, overall survival time for patients with low <i>KLK4</i> (n = 25) and high <i>KLK4</i> (n = 13) levels (χ² = 4.9, <i>p</i> = 0.03).</p

KLK4 induced uPA expression in SKOV-3 cells.

<p><b>A.</b> Western blot analysis shows expression of uPA, α5 integrin (ITN) and KLK4 (V5) of 3 KLK4 and 3 vector control clones with native SKOV-3 cells as a control with GAPDH as a loading control. <b>B.</b> Western blot analysis shows expression of KLK4 and uPA in KLK4-1 clone transfected with siRNA KLK4 exon 1 (psilK4Ex1), and both KLK4 exon 1 and 2 knockdown constructs (psilK4Ex1+2), p-silencing scramble (psil Ctl) and mock controls. GAPDH was used as a loading control. <b>C.</b> Western blotting shows expression of KLK4 and uPA in KLK4-1 cells cultured as 2D-monolayers (2D), 3D-collagen I (Col I), 3D-Matrigel (Matrigel), and 3D-suspension (Susp), with GAPDH as a loading control. <b>D.</b> Western blot shows expression of KLK4 and uPA in serous EOC cells of primary tumors (T) and ascites (A) from 6 patients. WCL of OVCA432 MCAs serves as a positive control and GAPDH as a loading control. <b>E.</b> Densitometry analysis of 3 Western blots indicative of that shown in <b>D.</b> **P<0.05 and ***P<0.001 indicate the significantly different levels of KLK4 and uPA in ascitic (A) and primary tumor cells (T).</p

MCAs clear mesothelial monolayers mimicking invasion into the peritoneal membrane.

<p><b>A.</b> Bright-field and fluorescence (CellTracker⁴⁹²) images show KLK4-1, Vector-1, SKOV-3 and OVCA432 MCA (4 h and day 3) clearance of mesothelial monolayers. Discontinuous lines indicate perimeters of the spreading MCAs. <b>B.</b> Quantitative analysis shows the average diameter of 10 MCAs from 3 separate experiments for above cell lines at 4 h and day 3 respectively; mean ± SE, n = 3, **P<0.01. <b>C.</b> IF microscopy images show mesothelial monolayer clearance of MCAs formed by KLK4-1 labeled with CellTracker⁴⁹², Vector-1, OVCA432 and SKOV-3 cells stained with an E-cadherin antibody (green); both MCAs and mesothelial LP9 cells were stained with Phalloidin for F-actin (Alexa Flour 568, red) and DAPI for nuclei (blue) respectively; discontinuous lines indicate positions of multiple Z sections shown as right and bottom panels. For panels <b>A</b> and <b>C</b>, scale bars, 50 µm.</p

Inhibition of KLK4 increased paclitaxel sensitivity.

<p>WST-1 assay shows cell survival of KLK4-1, KLK4-2, KLK4-3, Vec-1, Vec-2 clones and native SKOV-3 MCAs after paclitaxel (<b>A</b>) treatment in 3D-suspension. <b>B.</b> Left panel, representative images of KLK4-1 MCAs on day 4 with mouse IgG and a functional KLK4 blocking antibody, PBS as a control and KLK4 selective inhibitor SFTI-FCQR (SFTI) as indicated. Right panel, representative images of OVCA432 MCAs on day 4 with PBS as a control and KLK4 selective inhibitor SFTI-FCQR (SFTI) or aprotinin as indicated. Scale bars, 200 µm. <b>C.</b> Cell survival determined by WST-1 assay after treatment with paclitaxel (Pac) on 3D-suspension cultured KLK4-1 clone and OVCA432 cells +/−1 µM SFTI or 5 µM aprotinin (Aprot). Experiments in panels <b>A</b> and <b>C</b> were repeated 3 times in triplicate, bars represent Mean ± SEM. Statistical significance indicated as *P<0.05, **P<0.01.</p