anti c iap1 primary antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: cIAP1 inhibitor of apoptosis is a tumor suppressor in Ewing sarcoma

doi: 10.1101/2025.06.27.661947

Figure Lengend Snippet: a) Workflow depicting a stepwise systems biology approach to identify DEGs regulated by EWSR1::FLI1, involved in regulation of cell differentiation, and associated with overall survival in a cohort of 196 EwS patients. Number of genes represent remaining candidates after each filtering step. b) Network of EWSR1::FLI1-regulated genes involved in regulation of cell differentiation. Genes are depicted as nodes (circles). Node outline color represent up-(green) or down-(red) regulation by EWSR1::FLI1. Node border width represent strength of regulation by EWSR1::FLI1 (thicker border represents higher the fold-change). Node size represents strength of association with overall survival in a 196 EwS patient cohort. Yellow nodes depict strong and significant association with overall survival ( P <0.01). Connecting lines show three types of interconnections between nodes: physical (red), pathway (blue), genetic (green). c) Kaplan-Meier survival analysis of 196 primary EwS patients stratified by quintile cIAP1 expression. Mantel-Haenszel test. DEG: differentially expressed genes; KD: knockdown. d) cIAP1 expression levels of EwS and 14 additional primary pediatric tumors or EwS morphological mimics. Data are represented as bar plots where horizontal bars represent mean and SEM. The number of samples per group ( n ) is given in parentheses. ARMS: alveolar rhabdomyosarcoma; ALL: acute lymphocytic leukemia; PNET: primary neuroectodermal tumors; AML: acute myeloid leukemia. e) Analysis of single-cell RNA-Seq data (GSE XYZ) for cIAP1 expression comparing EwS patient derived xenografts (PDX) to mesenchymal stem cells (MSC). Rectangles depict EWSR1::FLI1 expression level (white: low; black: high). Number of analyzed cells is given in parentheses. f) cIAP1 and EWSR1::ETS expression in 18 EwS cell lines with conditional knockdown (KD) of EWSR1::FLI1 (gray dots) or EWSR1::ERG (black dots) for 96 h measured by Affymetrix microarrays and qRT-PCR, respectively. n = 3 biologically independent experiments. g) Relative cIAP1 expression as measured by qRT-PCR of TC-71 and SK-N-MC cells containing a DOX-inducible re-expression construct for cIAP1. n ≥8 biologically independent experiments. Two-sided Mann-Whitney test. h) Size-proportional Venn diagrams of genes concordantly regulated 96 h after KD of EWSR1::FLI1 or upregulation of cIAP1 in TC-71 and SK-N-MC EwS cells. Minimum log 2 fold-change ± 0.5. Fischer’s exact test. i) Weighted Gene Correlation Network Analysis (WGCNA) depicting functional gene enrichment of down- or up-regulated genes in cIAP1 re-expressing EwS cells. Networks depict signatures presenting P <0.05, NES>1.25. NES, normalized enrichment score. Arrows depict direction of gene regulation. j) WGCNA of protein-sets obtained by Pearson correlation analysis of proteins whose expression correlate with cIAP1 protein expression in four EwS cell lines present in the proteomic dataset from the Cancer Cell Line Encyclopedia (CCLE). Networks depict signatures presenting P <0,05, NES>2. k) Viable cell count of TC-71 and SK-N-MC cells containing a DOX-inducible re-expression construct for cIAP1 72 h after treatment with or without DOX. Data are mean and SEM, n =6 biologically independent experiments. Two-sided Mann-Whitney test. l) Relative colony formation as measured in clonogenic index of TC-71 and SK-N-MC cells containing a DOX-inducible re-expression construct for cIAP1 . Cells were grown either with or without DOX. n ≥6 biologically independent experiments. Two-sided Mann-Whitney test. Data are mean and SEM. m) Relative percentage of area covered by colonies grown in soft-agar of TC-71 and SK-N-MC cells containing a DOX-inducible re-expression construct for cIAP1 . Cells were grown either with or without DOX. n =4 biologically independent experiments. Two-sided Mann-Whitney test. n) Growth of EwS subcutaneous xenografts of TC-71 and SK-N-MC cells containing a DOX-inducible re-expression construct for cIAP1 (arrow indicates start of DOX-treatment). Data are represented as means ( n ≥5 animals/group). Two-sided Mann-Whitney test. o) Ex vivo analysis of tumor weight (left) and representative H&E images of tumors from animals treated without or with DOX (right), scale bar = 5mm. p) Ex vivo analysis of mitotic index of xenografted TC-71 and SK-N-MC cell lines. Data are mean, n ≥5 animals/group. q) Ex vivo analysis of Ki-67 positivity of xenografted TC-71 and SK-N-MC cell lines. Horizontal bars represent means and whiskers SEM, n ≥5 animals/group. P -values were determined via χ 2 test testing all positives (high and moderate immunoreactivity) versus negatives. Histological images depict representative Ki-67 micrographs. Scale bar=50μm. r) Ex vivo analysis of necrotic area of xenografted TC-71 and SK-N-MC cell lines upon cIAP1 re-expression. Two-sided Mann-Whitney test. s) Graph depicts mean number of apoptotic cells per high power field (HPF) of n ≥5 tumors per group analyzed.

Article Snippet: Mouse monoclonal anti-cIAP1 antibody (1:500 sc-271419, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or rabbit monoclonal anti-GAPDH antibody (1:1,000, #2118, Cell Signaling Technology Europe B.V. Leiden, The Netherlands) were used as primary antibodies.

Techniques: Cell Differentiation, Expressing, Knockdown, RNA Sequencing, Derivative Assay, Quantitative RT-PCR, Construct, MANN-WHITNEY, Functional Assay, Cell Counting, Ex Vivo