Both agents increased p21 levels whereas only the combination of TSA treatment with SBLC silencing failed to further increase p21 levels (Supplementary Figure S5B)

Both agents increased p21 levels whereas only the combination of TSA treatment with SBLC silencing failed to further increase p21 levels (Supplementary Figure S5B). are dependent on SENEBLOC expression. INTRODUCTION Cell senescence was described by Hayflick as a concept accounting for the finite lifespan of non-transformed fibroblasts (1). Senescence involves cells entering an essentially irreversible non-proliferative but nonetheless viable state. Characteristics of senescent cells include an enlarged size (1), resistance to IWR-1-endo apoptosis (2), changes in metabolic phenotype (3) the acquisition of senescence-associated heterochromatin foci (SAHF) (4), senescence-associated -galactosidase (SA–gal) activity (5) and the senescence-associated secretory phenotype (SASP) (6). Senescence is proposed as a defense mechanism to mitigate cancer development through preventing the replication of damaged genomes (7,8). Senescence also contributes to the age-related decline in organ function through the loss of progenitors and the accumulation of senescent cells (9,10). Broadly, there is replicative senescence (RS) involving the telomere length-dependent limit of cell divisions or stress-induced premature senescence which occurs independently of telomere erosion (11,12). Nevertheless, both forms involve sustained repression of pro-proliferative genes regulated through the retinoblastoma (Rb) pocket proteins to Tmem34 induce cell-cycle arrest. Senescence programming is principally achieved by activation of tumor suppressor networks encompassing p53/p21CIP1 and p16INK4a/ARF and is typified by increased levels of cyclin-dependent kinase inhibitors, p21 and p16 (8,10). Moreover, radiation and chemotherapy induce senescence in tumors, indicative that cancer cells possess the latent ability to undergo senescence (13,14). Of interest, the IWR-1-endo inactivation of c-Myc in cancer cells can also trigger senescence (15) and in melanoma, c-Myc can suppress oncogene-induced senescence (OIS) induced by activated forms of Braf and IWR-1-endo N-Ras (16). Although drivers of senescence are well accepted, the underlying control mechanisms are not fully understood. It has recently emerged that long non-coding RNAs (lncRNAs) play important regulatory roles (17,18). For example, the lncRNA PANDA is co-induced with p21 by DNA damage and serves to prevent transactivation of proliferative genes during senescence (19). The lncRNA HOTAIR increases during replicative and irradiation-induced senescence (20) and reducing the levels of lncRNA MALAT in cycling cells also induces senescence, an effect attributed in part to p53 activation (21). Thus, lncRNAs play positive and negative roles in senescence. The role of senescence in aging has given rise to the notion of senolytics, therapeutics that selectively remove senescent cells to prevent or reverse organ deterioration (9,14). Indeed such agents can re-sensitize senescent cells to apoptosis for example, the tyrosine kinase inhibitor, dasatinib can induce apoptosis in senescent adipocytes but not their non-senescent counterparts (22). The activation of mTOR signaling during senescence has been shown to promote the SASP and this is counteracted by rapamycin (23,24). Nevertheless, the mechanistic actions of rapamycin appear multifactorial (25). Here we describe SENEBLOC, a lncRNA that maintains normal and transformed cells in the non-senescent state. Under steady state conditions, SENEBLOC acts pleiotropically to repress p21 expression through both p53-dependent and independent mechanisms. SENEBLOC serves as a scaffold to facilitate p53-MDM2 association which decreases p53-dependent transactivation of p21. Alternatively, SENEBLOC acts as a decoy to sequester miR-3175 and prevent HDAC5 mRNA turnover which also contributes to p21 repression. Additionally, we show that the antagonistic actions of rapamycin on p21 expression are dependent on SENEBLOC. Moreover, we show that manipulating SENEBLOC in cancer cells has a profound growth effect. MATERIALS AND METHODS Cell culture HCT116, A549, IMR90, HAFF, 293T and P493-6 cells carrying a c-Myc tet-off system were maintained as previously described including mycoplasma testing and cell line authentication (26). Antibodies and reagents Supplementary Tables S3 and 4. Western blotting Equal amounts of protein or IWR-1-endo IP eluates were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes before immunodetection using ECL as previously described (26). RNAi Lentiviral supernatants were prepared in HEK293T cells after transfecting with shRNAs (cloned in PLKO.1; Supplementary Tables S5 and 6), gag/pol, rev and vsvg plasmids at the ratio of 2:2:2:1. Cell free culture supernatants were used to infect cells for 24 h before selection with puromycin (8?g/ml). PCR One microgram of total RNA isolated using TRIzol reagent (Invitrogen) was used to synthesize cDNA using the PrimeScript RT Reagent Kit (Takara). Quantitative polymerase chain reaction (qPCR) was performed using SYBR Green real\time PCR analysis (Takara) with the specified primers (Supplementary Table S7). PCR results, recorded as cycle threshold (Ct), were normalized against an internal control (\actin). Alternatively, RT-PCR was performed using Taq DNA polymerase (Vazyme). Standard curves assays were utilized where absolute quantitation of RNA expression was required. Plasmids containing the target cDNA of interest were used to construct standard curves by plotting Ct values against copy number as determined from the adjusted final concentration of plasmids. RNA.