HPV16 oncoproteins down-regulate RECK and TIMP-2 in HFKs.

<p><i>A,</i> Monolayer cultures of primary HFKs were transduced with pLXSN-based retroviral vectors expressing HPV16 E6wt and/or E7wt. RECK levels were determined by Western blot. Beta-actin was used as loading control. FF287 cell line was used as a positive control. <i>B,</i> Immunofluorescence detection of RECK (<i>green</i>) in HFKs expressing HPV oncoproteins. RECK is clearly detected in control and HPV16 E6wt expressing HFKs, while E7wt and E6E7-expressing cells exhibit reduced protein levels (<i>arrows</i>). Nuclei were counterstained with DAPI (<i>blue</i>). Original magnification 1000×. <i>C,</i> TIMP-2 was determined by ELISA (Biotrak). <i>P<0.05</i>.</p

RECK is down-regulated in CIN II/III and invasive carcinoma samples.

<p>Representative immunoreactivity of RECK in (A) Cervicitis, (B) cervical intraepithelial neoplasia II (CIN II), (C) cervical intraepithelial neoplasia III (CIN III) and (D) cervical invasive carcinoma. Note the strong RECK membrane staining pattern in cervicitis (black arrow), a diminished cytoplasmastic staining in CIN II/III and a very weak RECK staining in invasive carcinoma. Inset in (D): Sample incubated in the absence of primary antibody.</p

RECK expression in cervicitis, CIN I, CIN II/III and invasive cervical carcinomas.

<p>n = 66;</p>*<p>Weak = no staining or faint staining;</p>**<p>Strong = moderate or strong staining.</p

Expression of HPV-16 E6/E7 down-regulates RECK in organotypic cultures.

<p><i>A,</i> Representative immunoreactivity of RECK in control and HPV16 E6E7 expressing rafts cultures. Note the strong RECK membrane staining pattern (black arrow) in control compared to the faint staining in HPV16 E6E7 expressing rafts cultures. <i>B,</i> Quantitative real-time PCR (qRT-PCR) of <i>RECK</i> expression in organotypic cultures. The relative expression levels were normalized to tubulin. Bars represent the means of triplicate experiments; <i>bars</i>, ±SE.</p

RKIP expression in cervical lesions.

<p>Cervical intraepithelial neoplasia (CIN); Low-grade squamous intraepithelial lesions (LSIL); high-grade squamous intraepithelial lesions (HSIL); squamous cell carcinomas (SCC); adenocarcinomas (AC); adenosquamous carcinomas (ASC).</p

Correlations between RKIP expression and cervical cancer patient’s clinical data.

*<p>Log-rank test determined by Kaplan-Meier method.</p

Role of RKIP in cervical cancer cells response to cisplatin.

<p><b>A)</b> Representative pictures of the nonlinear regression analysis of cervical cancer cell lines treated with cisplatin for determination of half maximal inhibitory concentrations (IC₅₀). <b>B)</b> Graphic representation of the mean IC₅₀ values for cisplatin in the cervical cancer cell lines. The transfected cells with shRKIP were less sensitive to cisplatin treatment in the three different cell lines. <b>C)</b> HeLa transfected cell lines were exposed to increasing concentrations of cisplatin by 24 hours. Cisplatin treatment induced ERK activation (p-ERK1/2) and apoptosis of the cells as evaluated by PARP (total and cleaded specific antibodies) and caspase-9 cleavage, mainly in the empty vector transfected cells. All the experiments were done in triplicate at least three times. Data in the panels A and B are represented as the mean ± SD.</p

<i>In vivo</i> role of RKIP in HeLa cells growth and angiogenesis.

<p><b>A)</b> Representative pictures (16× magnification) of CAM assay after 7 days of tumor growth <i>in ovo</i> and <i>ex ovo</i>. <b>B)</b> Tumor growth was measured <i>in vivo</i> by CAM assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059104#s2" target="_blank">materials and methods</a> section. It was observed a higher perimeter (µM) of the tumors (<i>in ovo</i>) formed by shRKIP cells, however the difference between the control cells was not significant. <b>C)</b> Hematoxylin-eosin staining of the paraffin embedded tumors showing the higher vascularization induced by RKIP inhibition. Representative pictures with 10× and 20× magnification are represented in the left panel. <b>D)</b> Counting of the blood vessels <i>ex ovo</i>, it was observed a statistically significant increase on the number of vessels recruited in the tumors formed by shRKIP cells when compared to the control. In total it was analyzed 20 eggs (10 were injected with empty vector and 10 with shRKIP cells). The data is represented as the mean ± SD and differences with a <i>p</i><0.05 on the Student’s t test were considered statistically significant (*).</p

Immunohistochemistry analysis of RKIP in cervical tissues.

<p><b>A)</b> Positive expression in a cervicitis. <b>B)</b> High-grade squamous intraepithelial lesion with negative expression. <b>C)</b> Adenocarcinoma tissue depicting positive expression. <b>D)</b> Negative adenocarcinoma (*) with adjacent normal cells (arrow) depicting positive staining. All the pictures were taken with at 200× magnification.</p

Effect of RKIP on ERK pathway activation in HeLa cells.

<p><b>A)</b> Immunocytochemistry analysis for RKIP in HeLa, SiHa and C-33A cells showing both nuclear and cytoplasmatic expression. <b>B)</b> For RKIP inhibition, the cell lines were stably transfected with a shRNA for RKIP and with the respective empty vector for control. RKIP expression levels were assessed by western blot. Further, the cells were stimulated with 10 ng/ml of EGF by 10 minutes and EGFR and ERK activation (phosphorylation) was assessed by western blot for phospho-ERK1/2 and phospho-EGFR expression, but no significant differences were observed. E: Empty vector; Sh: ShRKIP.</p