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This revealed that SSRP1 mRNA, whereas showing significant variability among different samples, was elevated in the majority of tumors as compared to tissue from patients with no disease or noncancer-related diseases ( Figure?4). Cultured cell lines included in the analysis had the highest average level of SSRP1 of any category ( Figure?4A), suggesting that in?vitro conditions either induce SSRP1 expression or select cells with elevated SSRP1. SPT16 mRNA was also elevated in Cobimetinib purchase tumors, but to a lesser extent than SSRP1 ( Figure?S7). This difference was consistent with our finding that SSRP1 mRNA and protein both increased in the process of HMEC transformation, whereas for SPT16, only protein (not mRNA) levels increased ( Figures Bumetanide 1B and 1C). This is most likely due to the demonstrated dependence of SPT16 protein levels on SSRP1 ( Safina et?al., 2013). Nevertheless, as for SSRP1 mRNA, a significant number of tumors with very high levels of SPT16 mRNA were observed among various types of cancer. As a more direct evaluation of FACT expression in a clinical setting, we performed immunohistochemistry (IHC) staining of SSRP1 on tissue microarrays (TMAs) containing primary and metastatic tumors of different types as well as matching normal tissue from 793 patients (see Experimental Procedures). Tumors on the TMAs included invasive breast ductal and lobular carcinoma, non-small-cell lung cancer (NSCLC), renal cell carcinoma (RCC), and prostatic, pancreatic ductal (PDA), and colorectal adenocarcinomas. SSRP1 staining was used to assess FACT levels based on the previously established strong correlation between SSRP1 and SPT16 protein levels (Garcia et?al., 2011). SSRP1 staining was scored using a semiquantitative system reflecting both the intensity of staining and the proportion of positive cells (see Experimental Procedures). On the TMAs, all cells in normal tissue samples were SSRP1 negative, with the exception of epithelial cells at the bottom of intestinal crypts (Figures 5A�C5C; Garcia et?al., 2011). Similarly, whereas tumor samples were frequently SSRP1 positive (see below), stromal cells present in the Alisertib solubility dmso sample, constituting the tumor microenvironment, were invariably SSRP1 negative (Figures 5A�C5C). The highest incidences of SSRP1-positive samples were observed in NSCLC (45%�C63%), PDA (59%) and colon adenocarcinoma (50%) (Figure?5D). In contrast, very few cases of prostatic adenocarcinoma and RCC were SSRP1 positive (