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Fig. 1 | BMC Genomics

Fig. 1

From: A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence

Fig. 1

Epithelial cancer evolution experiment (ECEE) in a CDC6-expressing non malignant epithelial model. a-b Generation and validation of the HBEC CDC6 Tet-ON cellular system. a Schematic representation of the lentiviral vectors (PLVX-Tet3G-BSD and PLVX-TRE3G-CDC6-BleoR) utilized for CDC6 transduction in HBECs (see details in “Methods” section). HBECs preserve their epithelial phenotype upon ectopic expression of hTERT and mutant CDK4 for immortalization. Inverted-phase contrast microscopy showed preservation of the epithelial morphology in HBECs upon transduction with lentiviruses, followed by treatment with antibiotics in order to isolate clones with inducible CDC6 over-expression. Scale bar: 10 μm. b Efficiency of CDC6 induction was confirmed both at protein (western blot) and mRNA (qRT-PCR) levels at the indicated time points. c Plot showing inverse relationship between proliferation rate, as measured by BrdU incorporation overnight, and senescent phenotype, as assessed by GL13 staining [53], during the time course of CDC6 induction. d Morphological and kinetic features observed by inverted-phase contrast microscopy (Scale bar: 20 μm), GL13 staining (Scale bar: 20 μm) and wound healing assay (Scale bar: 80 μm) at specific time points of ECEE representing normal, precancerous and cancerous stages of tumorigenesis. Non-induced cells (“OFF”) are near normal, 6-day induced cells recapitulate precancerous lesions, where senescent cells are evident (dark orange cells in cartoon [s]), and cells that have escaped from senescence (“ESCAPED”) share the invasive characteristics of cancer cells (dark green cells in cartoon [i]). A continuous counter-interaction between the oncogenic acting force and the anti-tumor reacting force (senescence) takes places at the precancerous stage, leading eventually to the prevalence of the tumor promoting effect, bypass of senescence and emergence of cancer cells [36]

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