After 20?min, luminescence was recorded on an Envision 2104 Multilabel plate reader (PerkinElmer). rendered cells resistant to TNF, while pharmacologic inhibition of NF\B HAMNO in and are increasingly identified as tumor suppressors and oncogenes [4, 7], whereas others such as serve as functional biomarkers [8, 9]. SMG7 is an RNA surveillance factor that functions together with up\frameshift (UPF) factors to deadenylate and degrade target RNAs [10, 11]. Several studies have highlighted SMG7 conversation with P53 as influencing cellular survival [12, 13]. SMG7 has also been identified in a large cohort to be associated with prostate cancer [14]. Our previous work identified in a whole\genome mutagenesis PTPRC screen against TNF, a pleiotropic cytokine that can induce extrinsic apoptosis [15]. TNF can induce cytotoxicity in tumors [16] but also plays a central role in NF\B activation and inflammation. Yet, the functional role of downstream targets of SMG7 with respect to TNF and tumor biology is usually poorly comprehended. In this study, we examined gene expression in cells and found that lncRNAs rather than PTC\made up of transcripts were preferentially overexpressed, indicating that SMG7 uniquely targets this class of transcripts. Further evaluation of the TNF pathway in these cells identified decreased CYLD tumor suppressor protein as the source of apoptosis resistance. CYLD is usually a negative regulator of NF\B that acts at the pathway branchpoint between apoptosis and NF\B activation. Accordingly, downregulation of CYLD in cells reduced caspase activity and promoted NF\B\mediated survival, while overexpression and NF\B pharmacological inhibition re\established TNF sensitivity. Strikingly, and expression showed a near\universal correlation in diverse human malignancy cell lines and clear cell renal cell carcinoma patient survival. We further examined noncoding RNAs as favored degradation targets of SMG7. Overexpression of two lncRNAs, and showed strong protection against TNF that increased further upon knockdown. is an oncogene identified in Burkitt’s lymphoma [17], while is a stress\induced transcript upregulated in response to apoptotic stimuli [18]. HAMNO Administration of TNF to 3D spheroids produced widespread cell death in parental cells, while spheroids showed compaction with viability. Nevertheless, pharmacological sensitization of HAMNO the NF\B pathway in both cell lines suppressed CYLD\ and identify SMG7 as a key molecular switch for cell survival in response to TNF. 2.?Methods 2.1. Cell lines and culture conditions MCF\7 (RRID: CVCL_0031), NIH 3T3 (RRID: CVCL_0594), and 293T (RRID: CVCL_0063) cells were acquired from ATCC (Manassas, VA, USA). MCF\7, NIH 3T3, 293T, and immortalized mouse fibroblasts (MF) cells were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% FBS superior (Biochrom, Berlin, Germany), 100?UmL?1 penicillin, and 100?gmL?1 streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37?C in a humidified atmosphere with 5% CO2. Morphology of all cell lines was constantly checked for conformity with ATCC’s specifications, and cells were regularly tested for mycoplasma. 2.2. Cell viability assays Unless stated otherwise, 3??103 cells were counted by a ViCell cell counter (Beckman Coulter, Brea, CA, USA), seeded in 96\well plates, and treated with the respective compounds as indicated. For doseCresponse curves, serial dilutions of respective compounds were prepared in 100?L medium and cells were added on top in 100?L medium. Cell viability was assessed by the addition of Resazurin (Sigma, St. Louis, MO, USA) to final concentration of 50?m, and fluorescence was measured 6?h later at 540?nm excitation/590?nm emission in a PerkinElmer Envision 2104 (PerkinElmer, Waltham, MA, USA) Multilabel plate reader. At least three wells per condition were averaged, and viability is usually presented as percentage relative to respective control. For growth analysis, 3??104 cells were seeded in.