MOESM1 of The availability of the embryonic TGF-β protein Nodal is dynamically regulated during glioblastoma multiforme tumorigenesis

Additional file 1: Figure S1. Nodal immunostaining changes along the development of the oncospheres. (a) Small sphere showing only cells that with a symmetrical Nodal distribution in the cytoplasm. (b) Large sphere comprised by cells with distinct Nodal distributions. (c–e) Nodal immunostaining (red) in OB1 stem cells placed at the top of oncospheres is localized to the cell membrane (green, phalloidin, asterisk). (f–h) Cells located at the lateral edge of oncospheres harboring different heights presented Nodal immunostaining symmetrically distributed in the cytoplasm of cells. (i–l) Cells directly attached to the substrate also presented a symmetrical distribution of Nodal. (e, h, k, l) Optical slices projected on the YZ axis showing a virtual reconstruction of the oncosphere

Analysis of SOX2 expression in human osteosarcoma tissue and cells directly isolated from patients.

<p><b>A</b>. Representative immunohistochemistry images showing expression of SOX2 in osteosarcoma tissues from four patients (OS1, OS2, OS6 and OS12). <b>B</b>. Western blot analysis of SOX2 expression in primary tumor cells directly isolated from the tumor sites of eight osteosarcoma patients. Cyclophilin B was used as loading control. <b>C</b>. Representative immunocytochemistry image showing SOX2 expression in cells (OS6) immediately after tumor tissue dissociation. Scale bar, 100μm. <b>D</b>. Quantification of SOX2-positive cells from immunofluorescence in freshly dissociated samples from 11 osteosarcoma patients. <b>E</b>. Immunofluorescence staining of osteoprotegerin in patient-derived osteosarcoma cells in culture. Scale bar = 50 μm. OPG, osteoprotegerin.</p

Effect of chemotherapeutic agents in osteosarcoma cells viability.

<p><b>A</b>. MTT analysis of osteosarcoma cells isolated from 18 PRE samples (from 18 different patients) and 10 POST samples (from 8 different patients, as samples from OS2 and OS3 were collected at two different time-points after chemotherapy). <b>B-D</b>. MTT analysis of osteosarcoma cells isolated from four patients (OS1, OS4, OS6 and OS14) and treated with vehicle or <b>B</b>. cisplatin, <b>C</b>. doxorubicin, or <b>D</b>. methotrexate for 72h. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, Mann-Whitney U test.</p

Analysis of SOX2 and OCT4 expression in osteosarcoma cells treated with chemotherapy agents.

<p><b>A</b>. Correlation between presence of metastasis in osteosarcoma patients and SOX2 expression in cells derived from their tumors. <b>B</b>. Western blot analysis of SOX2 and OCT4 expression in primary tumor cells directly isolated from osteosarcoma patients (OS1 and OS6) before (PRE) and after (POST) chemotherapy treatments. Cyclophilin B was used as loading control. <b>C</b>. Western blot analysis of SOX2 and OCT4 expression in primary tumor cells (OS1 and OS9) after <i>in vitro</i> treatment with cisplatin, doxorubicin or methotrexate. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, Mann-Whitney U test.</p

Osteosarcoma patients’ characteristics, tumor topography, evolution, and surgical procedure.

<p>Osteosarcoma patients’ characteristics, tumor topography, evolution, and surgical procedure.</p

Effect of chemotherapeutic agents in SSEA4 expression in osteosarcoma cells.

<p><b>A, B</b>. Flow cytometry analysis (<b>A</b>) and quantification (<b>B</b>) of CD133, CD15 and SSEA4 expression in patient-derived osteosarcoma cells. <b>C, D</b>. Flow cytometry analysis (<b>C</b>) and quantification (<b>D</b>) of SSEA4 expression in primary osteosarcoma cells treated with cisplatin, doxorubicin or methotrexate. <b>E</b>. Bioluminescent imaging of mice injected with luciferase-expressing SaOs2 osteosarcoma cells and treated with cisplatin and doxorubicin (Cis+Dox), or cisplatin, doxorubicin and methotrexate (Cis+Dox+Mtx). <b>F</b>. Quantification of total flux from tumors. <b>G, H</b>. Flow cytometry analysis (<b>G</b>) and quantification (<b>H</b>) of SSEA4 expression in osteosarcoma cells isolated from tumors treated with cisplatin and doxorubicin (Cis+Dox), or cisplatin, doxorubicin and methotrexate (Cis+Dox+Mtx). * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, Mann-Whitney U test.</p

Antiproliferative Activity of <i>trans-</i>Avicennol from <i>Zanthoxylum chiloperone</i> var. <i>angustifolium</i> against Human Cancer Stem Cells

<i>Zanthoxylum chiloperone</i> var. <i>angustifolium</i> root bark was studied with the aim of finding novel molecules able to overcome cancer stem cell chemoresistance. Purification of a methanol-soluble extract resulted in the isolation of a known pyranocoumarin, <i>trans</i>-avicennol (<b>1</b>). Compound <b>1</b> demonstrated antiproliferative activity on glioma-initiating cells, whereas it was inactive on human neural stem cells. <i>trans</i>-Avicennol (<b>1</b>) activated the MAPK/ERK pathway and was also evaluated for its ability to inhibit the enzyme indoleamine-2,3-dioxygenase

Dose-response curves for compounds exhibiting the strongest cytotoxic effect on GSCs.

<p>(A) Chemical structures of selected compounds and dose-response curves of suloctidil (left), zuclopenthixol HCl (middle) and bisacodyl (right) with representative activity profiles on proliferating (─) and quiescent (▲) TG1 GSCs. The fitted sigmoidal logistic curve to ATP-Glo cell survival assay readouts is shown on each plot (n = 3). (B) Dose-response curves of suloctidil (left), zuclopenthixol HCl (middle) and bisacodyl (right) on proliferating TG1 cells (─), quiescent TG1 cells (▲), human primary astrocytes (О) and human fetal neural stem cells (◻). Curves were fitted as in A (n = 3). (C-D) Cell viability measurements (trypan blue staining) on proliferating (P) or quiescent (Q) TG1 cells (C) and HA cells (D) treated with bisacodyl (compound <b>1</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134793#pone.0134793.g009" target="_blank">Fig 9</a>).</p

Expression of stemness, pluripotency or differentiation related genes in TG1 and OB1 cells under different <i>in vitro</i> culture conditions.

<p>(A-B) Histograms representing mRNA expression levels of stemness, pluripotency or differentiation associated genes included in the TaqMan Human Stem Cell Pluripotency Array from Life Technologies in proliferating (P) and quiescent (Q) (9 days without medium renewal) TG1 (A) and OB1 (B) GSCs. Results were normalized to the 18S rRNA levels and expressed as ΔΔCt taking proliferating TG1 (A) and OB1 (B) GSCs as calibrator samples. Data are from three independent experiments, each performed in duplicate. (C-D) Histograms representing mRNA expression levels of Nanog, GBX2 and IFITM1 genes in proliferating (P) and quiescent (Q) TG1 (C) and OB1 (D) GSCs obtained after 9 days without medium renewal or quiescent cells after 9 days without medium renewal reintroduced in proliferating culture medium for 1–4 days (Q+1-Q+4). Results are expressed as in A-B. Data are from two independent experiments, each performed in duplicate.</p

Cell cycle analysis and Ki-67 expression in TG1 and OB1 GSCs under different <i>in vitro</i> culture conditions.

<p>(A) TG1 (upper panel) and OB1 (lower panel) GSCs maintained in culture for 1, 9 and 14 days without medium renewal (d1, d9, d14, respectively) or cells obtained after 9 days without medium renewal and subjected to freshly prepared culture medium for 5 days (Q+5) were permeabilized and stained with anti-Ki-67 antibodies conjugated to FITC and propidium iodide (PI). (B-E) Histograms represent the percentage of TG1 (B, C) and OB1 (D, E) cells present in the G1, S, G2/M phases of the cell cycle as well as the percentage of quiescent cells in G0 and of cells showing DNA fragmentation (sub-G1). Cells in the G2/M phase were further distinguished as a function of the Ki-67 proliferation marker expression levels (G2/M high Ki-67 and G2/M low Ki-67). Analysis was performed on cells maintained for 1–14 days without medium renewal (B, D for TG1 and OB1 cells, respectively) or on cells left without medium renewal for 9 days (Q) and re-introduced into freshly prepared culture medium for 1 (Q+1), 2 (Q+2), 3 (Q+3), 4 (Q+4) or 5 (Q+5) days (C, E for TG1 and OB1 cells, respectively).</p