CSCs treated with sFRP4 and temozolomide possess lower tumorigenic potential <i>in vivo</i>.

<p>Average tumor size (a, b) after subcutaneous injection of U87 CSCs treated with control (U), S, T, or S+T (* p value <0.05, ** p value <0.01, n = 4).</p

sFRP4 and temozolomide treatment of CSCs leads to the down-regulation of invasiveness, migratory, and colony forming potential.

<p>a) Micrograph images of soft agar colony assay of U87 CSCs after treatment with control, S, T, or S+T. Staining with crystal violet indicated the colony size (scale bar = 50μm). b) Angiogenic assay of U87 CSCs (treated for 24h with control or S, T, or S+T) seeded on a pro-angiogenic ECMatrix gel pre-coated plate. Staining with crystal violet detected tube formation and initial processes (scale bar = 100μm). c) After treating U87 CSC with control, S, T, or S+T for 24 hr, an <i>in vitro</i> transwell migration assay was performed. Crystal violet staining indicated cell migration across the transwell chamber (scale bar = 100μm).</p

Fura-2 calcium assay.

<p>Intracellular calcium assay determined by fluorescent radiometric Ca₂⁺ indicator Fura-2, in U87 CSCs treated with control, S, T, or S+T (* p value <0.05, ** p value <0.01, n = 3).</p

Inhibition of GBM CSCs by sFRP4 and temozolomide.

<p>a) and b) Graphs represent inhibition of U87 and U373 CSCs respectively after treatment for 24h with sFRP4- 250pg/mL (S), temozolomide- 25μM (T), and sFRP4+temozolomide (S+T). Results are the mean ± SD of three independent experiments performed in triplicates (* p value <0.05, ** p value <0.01, n = 3). c) Photomicrographs of sphere forming assay and immunocytochemical staining with CD133 after treatment with control, S, T, and S+T. Nuclei were counterstained with DAPI (blue), (scale bar = 100μm).</p

JC-1 assay.

<p>JC-1 mitochondrial depolarization assay after control, S, T, or S+T treatment of U87 and U373 CSCs.</p

Additional file 2: Figure S1. of Epigenetic reprogramming converts human Wharton’s jelly mesenchymal stem cells into functional cardiomyocytes by differential regulation of Wnt mediators

Showing characterization of WJMSCs. A1 Photomicrograph of a confluent layer of MSCs obtained from Wharton’s jelly (scale bar = 200 μm, n = 3). A2 Immunohistochemical staining of WJMSCs with vimentin and nuclei counter-stained with DAPI (scale bar = 100 μm, n = 3). A3 Quantitative RT-PCR of MSC marker CD44 and pluripotency markers Oct4, Nanog, and Sox2, and negative CD34 mRNA expression of WJMSCs (*p < 0.05, **p < 0.01, n = 3). B Flow cytometric analysis of WJMSCs for MSC-positive CD markers CD73, CD90, and CD105, and negative marker CD34. C1–C3 Trilineage differentiation of WJMSCs: Oil Red ‘O’ staining for adipocyte differentiation (C1), Von Kossa staining for osteocyte differentiation (C2), and Alcian Blue staining for chondrocyte differentiation of WJMSCs (C3) (scale bar = 100 μm, n = 3). (TIFF 9437 kb

Analysis of cancer stem cell spheroids from U87 and U373 cell lines for expression of the CSC marker CD133 by immunocytochemistry, semi-quantitative and quantitative RT-PCR, immunoblot, and flow cytometry.

<p>a) Photomicrographs of monolayer and spheroid colonies (left panel, scale bar = 50μm, n = 3) and CD133 marker staining (right panel, scale bar = 100μm) as shown by immunocytochemistry in monolayer and spheroid colonies of U87 and U373 cell lines. b) Quantitative RT-PCR of CD133 mRNA expression of U87 and U373 cell lines grown in CSC medium. Results are the mean ± SD of three independent experiments performed in triplicates (* p value <0.05, ** p value <0.01, n = 3). ML = monolayer, CSC = cancer stem cells. c) Flow cytometry analysis of CD133 in U87 and U373 cells grown in CSC medium.</p

Additional file 1: Table S1. of Epigenetic reprogramming converts human Wharton’s jelly mesenchymal stem cells into functional cardiomyocytes by differential regulation of Wnt mediators

Presenting cardiac-specific gene primers, Table S2 presenting Wnt-related gene primers, Table S3 presenting stemness and noncardiac gene primers, and Table S4 presenting methylation-specific primers. (DOCX 20 kb

Primer sequences for CSC marker, EMT and drug resistance associated genes.

<p>Primer sequences for CSC marker, EMT and drug resistance associated genes.</p

Treatment of CSCs with sFRP4 results in down-regulation of functional mesenchymal protein expression.

<p>a) Immunocytochemistry of mesenchymal markers α smooth muscle actin (α SMA), vimentin, and ß-catenin in U87 CSCs treated with control, S, T, or S+T (scale bar = 100μm). Nuclei were counterstained with DAPI (blue). b) Immunoblot analysis of α SMA, vimentin, and ß-catenin in U87 CSCs treated with control, S, T, or S+T.</p