Journal: Journal of Cellular and Molecular Medicine

Article Title: Endothelial cell differentiation of human breast tumour stem/progenitor cells

doi: 10.1111/j.1582-4934.2008.00338.x

Figure Lengend Snippet: Mammosphere generation, characterization and epithelial differentiation. Mammospheres were obtained by culture of dissociated cells from a human breast tumour in mammosphere medium containing EGF and bFGF. ( A ) Representative micrograph showing the ematoxilin and eosin staining of paraffin-embedded, formalin-fixed mammospheres. ( B ) Mammosphere clone formation analysis in secondary and tertiary passages. The number of mammosphere clones/100 cells generated from single cells increased in the third passage. ( C ) Representative FACS analysis of mammosphere cell clones showing the expression of CD44 and not CD24 and of VEGF receptor 1 (VEGFR1; the dark area indicates the specific Ab, the white area the isotypic control). ( D ) Representative immunofluorescence expression by mammosphere cell clones of the stem cell markers Oct-4 and nestin, but not of the differentiation markers CK14, CK18 and α-SMA. ( E ) Serum supplementation and withdrawal of growth factors induced the expression of lineage specific markers of the mature mammary epithelium (CK14, CK18 and α-SMA) in cultured cells from mammosphere clones, with loss of the stem cell markers Oct-4 and nestin. ( F ) Representative FACS analysis of epithelial differentiated cells showing the acquirement of CD24, the expression of pan-CK and CK18 and the absence of the endothelial cell markers KDR and CD31. The dark area indicates the specific Ab, the white area the isotypic control. Original magnification: panel A: ×100; panel D and E: ×650. Nuclei were counterstained with Hoechst dye.

Article Snippet: The following antibodies were used: rabbit anti-von Willebrand Factor (vWF) Ab and anti-α-smooth muscle actin (α-SMA) mAb (Dako), rabbit anti-pan-CK Ab, goat anti-CK14, rabbit anti-CK18 Abs (Santa Cruz), anti-vimentin mAb (Sigma, clone 13.2), goat anti-Oct-4 Ab (Abcam, Cambridge, UK) and rabbit anti-nestin Ab (Chemicon, Temecula, CA, USA).

Techniques: Staining, Clone Assay, Generated, Expressing, Control, Immunofluorescence, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Endothelial cell differentiation of human breast tumour stem/progenitor cells

doi: 10.1111/j.1582-4934.2008.00338.x

Figure Lengend Snippet: Endothelial differentiation of mammosphere clones cultured in endothelial differentiating medium containing VEGF. ( A ) Representative micrographs showing the expression of endothelial markers after 2-week culture by immunofluorescence staining of vascular endothelial (VE)-cadherin and vWF, and representative FACS analysis (KDR, CD105, CD31, CD146, VEGF receptor 3 (VEGFR3): black area; the white area is the isotypic control) showing the endothelial differentiation. CK18 was negative. ( B ) Representative micrographs showing the formation of capillary-like structures on Matrigel by endothelial-differentiated breast tumour stem cells, but not by undifferentiated breast tumour progenitor cells. Capillary-like formation assay was evaluated after 6 hrs. (Original magnification: panel A ×650; panel B ×200).

Article Snippet: The following antibodies were used: rabbit anti-von Willebrand Factor (vWF) Ab and anti-α-smooth muscle actin (α-SMA) mAb (Dako), rabbit anti-pan-CK Ab, goat anti-CK14, rabbit anti-CK18 Abs (Santa Cruz), anti-vimentin mAb (Sigma, clone 13.2), goat anti-Oct-4 Ab (Abcam, Cambridge, UK) and rabbit anti-nestin Ab (Chemicon, Temecula, CA, USA).

Techniques: Clone Assay, Cell Culture, Expressing, Immunofluorescence, Staining, Control, Tube Formation Assay

Journal: PLoS ONE

Article Title: The Use of a New CellCollector to Isolate Circulating Tumor Cells from the Blood of Patients with Different Stages of Prostate Cancer and Clinical Outcomes - A Proof-of-Concept Study

doi: 10.1371/journal.pone.0158354

Figure Lengend Snippet: The CTCs were identified and enumerated via a) positive nuclear staining (Hoechst), b) positive cytokeratin staining, and c) negative CD45 staining. d) Overlay of all images showing size and morphological characteristics.

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin (BSA) (PAA) in PBS for 1 h. Primary antibodies, including anti-pan-cytokeratin-fluorescein isothiocyanate (FITC) (CK8, CK18 and CK19, Abcam) and anti-CD45- allophycocyanin (APC, Invitrogen), were added for 1 h. The wire was then rinsed 3 times with 3 ml of PBS, and the nuclei were counterstained with Hoechst 33258 (Invitrogen).

Techniques: Staining

Journal: PLoS ONE

Article Title: Microenvironment Promotes Tumor Cell Reprogramming in Human Breast Cancer Cell Lines

doi: 10.1371/journal.pone.0083770

Figure Lengend Snippet: (A) Representative western blot showing the levels of the proteins E-cadherin and β-catenin in MDA-MB-231 cells, after 7 and 11 days of treatment with EW. Alpha tubulin was used as a loading control. At the right of the panel are reported the densitometric quantification of E-cadherin and β-catenin respectively, obtained normalizing the O.D. of protein bands versus the O.D. of α-tubulin bands, chosen as loading control. The results are the mean (SD) of three independent experiments; *p<0.001 and # p<0.05 vs ctrl by Student’s t test. (B) Confocal microscopy of one representative optical section of MDA-MB-231 cells at the seventh experimental day: the immunostaining for β-catenin (green) shows a dispersed and punctuated staining in control condition (ctrl) with respect to the stronger staining that localized near the membrane perimeter for EW-treated cells. (C) Representative western blot showing the levels of the proteins CK18 and α-SMA in MDA-MB-231 cells, after 5 days of treatment with EW. Alpha tubulin was used as a loading control.

Article Snippet: Blotted proteins were probed with the following antibodies: anti-β-casein (sc-30041), anti-β-catenin (sc-7963), anti-E-cadherin (sc-7870), anti-oct-3/4 (sc-5279), anti-ZEB1 (sc-10570), anti-ZEB2 (sc-18392), and anti-c-myc (sc-40) all from Santa Cruz Biotechnology; anti-α-tubulin (T5168) from Sigma; the anti-Histone H3 trimethyl Lys 4 antibody (39159) from Active Motif; the anti-Histone H3 antibody (ab1791), anti-klf4 (ab75486), anti-sox2 (ab97959), anti-nanog (ab80892) and the anti- α-SMA (ab5694) were from Abcam; the anti-LSD1(#2139), anti-Snail (#3879) and anti-CK18 (#4548) antibodies were from Cell Signaling.

Techniques: Western Blot, Control, Confocal Microscopy, Immunostaining, Staining, Membrane

Journal: PLoS ONE

Article Title: Microenvironment Promotes Tumor Cell Reprogramming in Human Breast Cancer Cell Lines

doi: 10.1371/journal.pone.0083770

Figure Lengend Snippet: Phase contrast microscopy. (A): confluent MCF-7 cells in OV condition (day 7). (B): confluent MDA-MB-231 cells in OV condition (day 7): OV-treated cells did not form acini or duct-like structures in both cell lines. (C) and (D): representative western blot showing the levels of the protein β-casein in MCF-7 and MDA-MB-231 cells respectively, after 5 days. (E) and (F): representative western blot showing the levels of the proteins E-cadherin, CK18, and α-SMA in MDA-MB-231 cells, after 5 days; α-tubulin was used as a loading control. Ctrl (control), EW (egg white), and OV (ovalbumin).

Article Snippet: Blotted proteins were probed with the following antibodies: anti-β-casein (sc-30041), anti-β-catenin (sc-7963), anti-E-cadherin (sc-7870), anti-oct-3/4 (sc-5279), anti-ZEB1 (sc-10570), anti-ZEB2 (sc-18392), and anti-c-myc (sc-40) all from Santa Cruz Biotechnology; anti-α-tubulin (T5168) from Sigma; the anti-Histone H3 trimethyl Lys 4 antibody (39159) from Active Motif; the anti-Histone H3 antibody (ab1791), anti-klf4 (ab75486), anti-sox2 (ab97959), anti-nanog (ab80892) and the anti- α-SMA (ab5694) were from Abcam; the anti-LSD1(#2139), anti-Snail (#3879) and anti-CK18 (#4548) antibodies were from Cell Signaling.

Techniques: Microscopy, Western Blot, Control