Figures S1-S7, Tables S1-S6, Supplementary Materials and Methods, Supplementary References from Tissue Tranglutaminase Regulates Interactions between Ovarian Cancer Stem Cells and the Tumor Niche

Abstract

<p>Supplementary Figure S1. ALDH1A1, Sox2, Nanog, Oct-4 and TG2 expression in OCSCs and ovarian spheroids. A-B. ALDH1A1, Sox2, Nanog, and Oct-4 mRNA levels measured in ALDH+/CD133+ vs. ALDH-/CD133- isolated from OVCAR5 and COV362 cells. C-D. Expression of TG2, integrin β1 (ITGB1), and FN1 in primary OC cells and in SKOV3 cells grown as monolayers (mono) or spheroids (sphere). E-F. ALDH1A1, Sox2, Nanog, and Oct-4 mRNA levels measured in monolayers (mono) vs. spheroids (sphere) from human primary OC cells and in SKOV3 cells. G. W.B. of TG2 expression levels in OC cell lines (SKOV3 and HEY) stably transduced with AS-TG2 or ShRNA targeting TG2 (Sh-TG2). H. Flow cytometry measures ALDEFLUOR-FITC+/CD133-APC+ in SKOV3 cells (pcDNA3.1 and AS-TG2) and HEY (Sh-Ctr and Sh-TG2) cell lines. Supplementary Figure S2. mRNA microarray comparison between TG2, FN1 and integrin β1 (ITGB1) (A-B) in the Affymetrix Human Exon 1.0 ST ovarian cancer TCGA database. C. Estimated overall survival curves for tumors expressing high levels of TG2 and of ITGB1 vs. those expressing low levels of TG2 and of ITGB1 vs. all others in the Affymetrix Human Exon 1.0 ST TCGA database. Supplementary Figure S3. Effects of anti-TG2 (4G3) treatment in OCSCs. A. Flow cytometry measures apoptosis and necrosis of OVCAR5, SKOV3, COV362 cells grown as spheroids and treated with 4G3. B-C. IF staining for TG2, FN, and integrin β1 in primary OC cells grown as spheroids after 4G3 treatment. Supplementary Figure S4. TG2/FN/Integrin β1 complex regulates stemness and β-catenin activation. A. ALDH1A1, Sox2, Nanog, and Oct-4 in 4G3 mRNA expression levels in 4G3 treated COV362 spheroids. B. Q-RT-PCR for β-catenin, c-Myc, and cyclin D1 in 4G3 treated COV362 spheroids. Supplementary Figure S5. Functions of the TG2/FN/Integrin β1 complex and Fzd7 in OC cells. A. Q-RT-PCR for Fzd1 and Fzd7 in COV362 CSCs compared to non-CSCs. B. TCF/LEF luciferase reporter in 4G3 treated COV362 cells plated as spheroids. C. IHC staining for β-catenin in tumors derived from xenografts. D-E. IF staining for integrin β1, FN, and Fzd7 in 4G3 treated OC cell lines. Supplementary Figure S6. TG2-targeting disrupts OC spheroids proliferation and response to Wnt ligand. A-D. Proliferation assay measures the number of cells growing as spheroids derived from OC cell lines (OVCAR5, COV362, and SKOV3) and from primary OC cells after treatment with 4G3 and Wnt7A. Supplementary Figure S7. Activation of Wnt pathway in OC spheroids. A. Q-RT-PCR for Fzd7 in SKOV3 cells stably transduced with scrambled- or Fzd7-targeting ShRNA. B-C. CCK8 assay measures proliferation of SKOV3 cells stably transduced with scrambled- or Fzd7-targeting ShRNA clones and treated or not with Wnt3A or Wnt7A. D-I. Q-RT-PCR measures c-Myc and cyclin D1 mRNA expression levels in OVCAR5 and SKOV3 cells stably transduced with scrambled- or Fzd7-targeting ShRNA in basal conditions or after Wnt3A/7A. J. TG2 mRNA expression level correlates with Fzd7 in the OC Affymetrix Human Exon 1.0 ST TCGA database. Supplementary Table S1. TG2/integrin β1 co-localization detected by PLA in human ovarian tumors. Supplementary Table S2. Univariate analysis overall survival Agilent 244K Gene Expression G4502A-07 data. Supplementary Table S3. Multivariate analysis overall survival Agilent data. Supplementary Table S4. Univariate analysis overall survival Affymetrix Human Exon 1.0 ST data. Supplementary Table S5. Multivariate analysis overall survival Affymetrix Human Exon 1.0 ST data. Supplementary Table S6: Primers used for Quantitative RT-PCR. Supplementary Materials and Methods. Supplementary References.</p>