The latter was not feasible until now due to inherited flaws of SA–Gal, the currently available method [40], whereas GL13 enables detection of senescent cells in any given biological material [53]. As a triggering oncogenic stimulus we favored the replication licensing factor CDC6 mainly because it is frequently over-expressed in various epithelial cancers from their earliest stages of development [60, 61]. In the cytoplasm processing of pre-miRs is mediated by Dicer, a class 3 RNase III, which along with various RBPs, including TRBP, stabilize Dicer. Dicer-TRBP complex liberates small RNA duplexes that are loaded onto Argonaute protein members (Ago1-4) forming effector complexes called pre-RISCs. Pre-RISCs remove the passenger miRs strand generating the mature form of RISCs encompassing single strand miRs (~22 nucleotides long each). The functional strand of miRs loaded on Ago1-4 guides RISCs to silence target mRNAs in the cytoplasm (C) through translational repression, mRNA cleavage and deadenylation [138]. Additionally, miRs may translocate into: a) the nucleus (N) [16], regulating the biogenesis of coding and non-coding RNAs (active RISC complexes are present in the nucleus (nRISC) having a distinct composition from cytoplasmic RISC (cRISC) [139]) and b) the mitochondria (M) bound to Ago2 at pre-RISC or mature RISC complex (mRISC) [17], regulating the translation of the mRNAs produced by mitochondrial genome which, in turn, modulate mitochondrial homeostasis [140]. Evidence also supports the presence of mitochondrial miRs encoded by mitochondrial genome [18]. A substantial fraction of miRs may also exist in the cytoplasm in an Ago-free form [141]. Notably, apart from DGCR8 and TRBP, different RBPs recognize distinct miR precursors regulating miR biogenesis [142]. (PDF 1025?kb) 12864_2017_4375_MOESM1_ESM.pdf (9.0M) GUID:?404ACC6E-FCEB-494F-AC8D-852CC3848CD5 Additional file 2: ITGB2 Table S1: Subcellular localization of miRs. (XLSX 12?kb) 12864_2017_4375_MOESM2_ESM.xlsx (13K) GUID:?DB412799-6560-4CA0-B6BA-6C66C2C8F4F2 Additional file 3: Table S2: Studies on miR expression in senescent cells. (XLSX 20?kb) 12864_2017_4375_MOESM3_ESM.xlsx (21K) GUID:?469526FC-32FB-47CD-908A-49D6A5DE30A7 Additional file 4: Table S3: Genes triggering oncogene-induced senescence. (XLSX 12?kb) 12864_2017_4375_MOESM4_ESM.xlsx (13K) GUID:?8D608B6B-B763-4495-AEF8-C4AB308934A2 Additional file 5: Figure S2: RB phosphorylation in HBEC CDC6 Tet-ON system. Immunoblot analysis of total and phosphorylated RB levels. CDK4 over-expression in HBEC results in continuous phorsphorylation of RB protein, while induction of CDC6 increased p-RB due to transcriptional down-regulation of p16 [63]. Actin serves as loading control. (PDF 21?kb) 12864_2017_4375_MOESM5_ESM.pdf (21K) GUID:?B4B30993-14C0-4363-ABB9-40EAD6DC2A41 Additional file 6: Figure S3: CDC6 binding onto the promoters of and loci of HBEC CDC6 Tet-ON system leading to transcriptional down-regulation. a) Chromatin immunoprecipitation (ChIP) assay showed that MYC-tagged CDC6 is bound on Vancomycin hydrochloride both the regulatory domain (RD) of locus and the Epal element of locus is enriched in DNA extracted from both anti-CDC6 (endogenous and exogenous) and Vancomycin hydrochloride anti-MYC-tag (exogenous) IPs in HBEC CDC6 over-expressing cells normalized to input and (RNA Pol II-IP serves as a negative control confirming transcriptional down-regulation). c) ChIP samples run on a SDS-PAGE gel revealed that CDC6 is accessible and immunoprecipitated by both CDC6 and MYC-tag antibodies with the protocol followed. (PDF 146?kb) 12864_2017_4375_MOESM6_ESM.pdf (480K) GUID:?CB8E164E-32EF-4BDA-B12D-496ECB36083A Additional file 7: Figure S4: Morphological features of HBEC CDC6 Tet-ON. a) Inverted-phase contrast photographs (Scale bar: 25?m) and bi) GL13 staining showed the dominance of senescent, flattened and multinucleated cells upon 6-day CDC6-induction; features that were substituted by a spindle morphology in the escaped cells. Traces of Vancomycin hydrochloride GL13 staining in the early “escaped” cells (indicated by arrows) prove their origin from senescent cells. bii) Sa–Gal activity correlates with GL13 staining. (Scale bar: 15?m). (PDF 689?kb) 12864_2017_4375_MOESM7_ESM.pdf (689K) GUID:?82173380-CAE4-4F2B-AA64-294F51BF6125 Additional file 8: Figure S5: Comparative Inverted DAPI Banding karyotyping of 20 metaphase spreads from the OFF (on the left) and the escaped (on the right) cells. Arrows indicate random chromosome rearrangements (chromosomal instability). The rates of random structural chromosome rearrangements were found 3.5-times more pronounced in the “escaped” cells. (PDF 554?kb) 12864_2017_4375_MOESM8_ESM.pdf (554K) GUID:?80D10234-AABE-40E7-B455-F50CF56F8ADB Additional file 9: Figure S6: Schematic presentation of an R loop. R loops are three-stranded nucleic acid structure. Factors (upper left corner) that promote R loops are indicated as well as the differential cellular effects (bottom) stemming from their formation. (PDF 557?kb) 12864_2017_4375_MOESM9_ESM.pdf (557K) GUID:?2FBD8B50-1383-4373-B9B9-9C7DE313C98E Additional file 10: Figure S7: Bedgraphs of indicative genes showing the specificity of RNAseq analysis. RNAseq data from two biological replicates is depicted. (PDF 47?kb) 12864_2017_4375_MOESM10_ESM.pdf (48K) GUID:?BDD2532C-7642-4067-B179-60BEAD1C64C9 Additional file 11: Table S4: A Upregulated genes in induced (ON) HBEC CDC6 Tet-ON cells. b Down regulated genes in induced (ON) HBEC CDC6 Tet-ON cells. c Upregulated genes in ESCAPED (ESC) HBEC CDC6 Tet-ON cells. d: Down regulated genes Vancomycin hydrochloride in ESCAPED (ESC) HBEC CDC6 Tet-ON cells. (XLSX 630?kb) 12864_2017_4375_MOESM11_ESM.xlsx (631K) GUID:?C504D996-8882-40BA-9ECD-9BB0B422956D Additional file 12: Figure S8: Enrichment plots a-b) of the Cell cycle mitotic and c-d) of the DNA replication gene-sets. Cells entering.