FHL2 silencing decreases osteosarcoma cell growth.

<p>After treatment with Wnt3a CM (A) or FGF-2 (0.50 ng/ml<b>)</b> (B) for 3 days, DNA replication was evaluated by BrdU incorporation in shControl and shFHL2-transduced K7M2 cells. Apoptosis was induced by serum deprivation and effector caspases activity was evaluated at 48 h in cells treated with or without Wnt3a CM (C). a: p<0.05 <i>vs</i> untreated, b:p<0.05 <i>vs</i> shControl. TUNEL analysis was performed in basal and serum deprivation conditions at 72 h (D). *: <i>P</i><0.05 <i>vs</i> the indicated group or shControl cells.</p

Basal FHL2 expression in human osteosarcoma cells and in tissue microarrays (TMA) of human osteosarcomas.

<p>Whole cell lysates were probed with the indicated antibody and revealed by Western blot analysis (A). FHL2 expression was determined by immunohistochemistry in tissue sections of normal bone, primary tumors, metastatic or recurrent osteosarcoma (Mag.×125) (B). Semi-quantitative scoring of immunohistochemical staining with anti-FHL2 antibody in normal bone and osteosarcoma samples according to patient outcome (primary tumor, metastatic or recurrent osteosarcoma) (C). *<i>P</i><0.05.</p

FHL2 silencing decreases bone tumor cell migration and invasion.

<p>Migration of shControl and shFHL2-transduced K7M2 cells was evaluated by Boyden’s chamber (A) and wounding assays (C) and migrating cell number was evaluated (B, D). K7M2 cell invasion was evaluated by the Matrigel invasion assay (E, F). *: <i>P</i><0.05 <i>vs</i> shControl-transduced cells.</p

FHL2 silencing reduces lung metastasis in mice.

<p>Histological hematoxylin/eosin (H&E) staining of lung tissue sections showing metastasis (stars) developed in mice injected IM with shControl or shFHL2-transduced K7M2 cells (A). Metastasis area (B) and number (C) in the lung tissue were evaluated. Results are expressed as mean ± s.d. (n = 9 animals per group). *<i>P</i><0.05 <i>vs</i> shControl. Proposed model in which FHL2 silencing using shFHL2 in murine osteosarcoma cells attenuates Wnt/β-catenin signaling and reduces the expression of Wnt5a and Wnt10b and possibly other FHL2 target genes in the tumors, resulting in decreased osteosarcoma cell growth, invasiveness and tumorigenesis <i>in vivo</i> (D).</p

FHL2 silencing decreases Wnt/β-catenin signaling in osteosarcoma cells.

<p>Cell lysates of osteoblast precursor cell (C3H10T1/2), calvaria-derived osteoblastic cells (MC3T3E1) and osteosarcoma cell lines (K7M2) were analysed by western blot and FHL2 level was corrected for β-actin (A). After transduction with shControl or shFHL2, FHL2 levels in K7M2 cells were evaluated by q-PCR (B) and Western blot analysis (C). shControl and shFHL2 transduced K7M2 cells were treated for 24 h with Wnt3a CM and β-catenin nuclear translocation in K7M2 cells was evaluated by Western blot analysis of nuclear fraction (D), immunocytochemistry (red, arrows: β-catenin, blue: DAPI) (E), and β-catenin transcriptional activity was determined by a reporter assay (F). The mRNA levels in the shControl and shFHL2 cells were evaluated by q-PCR analysis (G, H). *: <i>P</i><0.05 <i>vs</i> the indicated group or shControl cells.</p

"Ich bin ein Sachse, protestiere aber nicht, wenn mich ein bodenständiger Deutscher für einen Rumänen hält." : das (inter-)kulturelle Portrait Paul Schusters

This article is dedicated to the intercultural aspects of Paul Schuster’s stories (1930-2004), a German writer, born in Sibiu, regarded by German literary historians and criticists as one of the most talented prose writers descending from the small German cultural enclave of Transylvania. His work is thematically focused on events of the past century; The German minority he belongs to plays a decisive role, but also its cohabitation with different ethnic groups in Romania as well as the interethnic relations between them. Interculturality in Paul Schuster's stories is revealed on several levels: cultural exchanges between different ethnic groups, aspects of interethnic collaboration, imagology, linguistic interferences and translations from Romanian authors

Additional file 8: Figure S7. of EPCR promotes breast cancer progression by altering SPOCK1/testican 1-mediated 3D growth

Cell growth kinetics of APC-stimulated breast cancer cell lines. A. MTS proliferation assay of cells stimulated with increasing doses of APC. Data were normalized with absorbance values from day 0. Each dot represents mean ± SD of six replicates. B. Percentage of cells in each phase of the cell cycle in control and 50 nM APC-stimulated cells for 24 and 48 h, in serum-free and 4% serum medium. C. Percentage of apoptotic cells in basal and staurosporine-induced conditions, measured by annexin-V binding flow cytometry assay. Cell lines are MDA-MB-231,1833, BT-549, and ANV5, from the left to the right, in all figure sections. (PPTX 325 kb

Additional file 3: Figure S2. of EPCR promotes breast cancer progression by altering SPOCK1/testican 1-mediated 3D growth

Effects of EPCR silencing in vitro and in vivo tumor growth in an orthotopic model. A. Western blot analysis of EPCR protein levels in human BT-549 (top) and murine ANV5 (bottom) cells transduced with a scramble shRNA (shControl) and shRNAs targeting human (shEPCR#1 and shEPCR#2 in BT-549) and murine (shEPCR#3 in ANV5) EPCR. β-tubulin was used as loading control. White line indicates that the membrane was cut. B. MTS in vitro proliferation assay of BT-549 (top) and ANV5 (bottom) cells. Data were normalized with absorbance values from day 0 and represent mean ± SD of six replicates. C. Percentage of BT549 (top) and ANV5 (bottom) cells in each phase of the cell cycle after maintaining cells in culture for 24 and 48 h. Sta, staurosporine. D. Percentage of apoptotic BT-549 (top) and ANV5 (bottom) cells in basal and staurosporine-induced conditions, measured by annexin-V binding flow cytometry assay. E. Quantification of spheres grown in 3D matrigel cultures. Data are mean ± SD of 8 replicates. Representative images at ×4 magnification. Scale bar 0.5 mm. F. Outline of the in vivo orthotopic experiment (n = 8 per group). G. Quantification of tumor volume at day 15 post-injection. H. Kaplan–Meier curves of resection-free survival. (PPTX 9850 kb

Additional file 4: Figure S3. of EPCR promotes breast cancer progression by altering SPOCK1/testican 1-mediated 3D growth

Immunohistochemical analysis of several markers in control and EPCR-silenced size-matched mammary tumors resected at different time points. A. Representative images showing H&E staining (×2.5 magnification) and the immunohistochemical staining of Ki67, cleaved caspase-3, CD31, and F4/80 (×20 magnification) in formaldehyde-fixed tumors. Scale bars 80 μm (H&E) and 10 μm (Ki67, caspase-3, CD31, and F4/80). T. mass, tumor mass. T. border, tumor border. B. Quantification of the percentage of immunoreactive cells. Each dot represents one tumor. Data are mean ± SEM. ns means non-statistical significance. (PPTX 2780 kb