Expression profiling by high throughput sequencing
Summary
Oncogene induced senescence (OIS) is a tumour suppressive response to oncogene activation that can be transmitted to neighbouring cells through secreted factors of the senescence associated secretory phenotype (SASP). Using single-cell transcriptomics we observed two distinct endpoints, a primary marked by Ras and a secondary by Notch. We find that secondary senescence in vitro and in vivo requires Notch, rather than SASP alone as previously thought. Currently, primary and secondary senescent cells are not thought of as functionally distinct endpoints. A blunted SASP response and the induction of fibrillar collagens in secondary senescence compared to OIS point towards a functional diversification.
Overall design
(Smart-seq2 Time course experiment): ER:RasG12V -expressing cells were induced into senescence for 7 days with 4-hydroxytamoxifen (4-OHT) and were isolated as single cells using FACS. Single cells that were not induced with 4-OHT were used as control. Single cells were also collected at another two time points, day 2 and day 4, before they became fully senescent on day 7. (10x Co-culture experiment): GFP cells are co-cultured with ER:Ras expressing cells and were induced into senescence with 4-OHT. Single cell 3' gene expression data was generated using 10x Genomics Chromium. (Smart-seq2 Hepatocyte experiment): Ex vivo primary hepatocytes were isolated using a modified retrograde perfusion technique as previously described[27]. Briefly, following perfusion with Perfusion Medium (Gibco) for 5 min, and Liver Digest Medium (Gibco) for 10 min, the liver was excised and the capsule disrupted to yield a cell suspension that was collected in Williams' E Medium supplemented with 2% FCS. Hepatocytes were purified by pelleting through a 40% (v:v) percoll gradient prior to FACS analysis. (10x dnMAML1 experiment): We used normal diploid human female lung fibroblasts IMR90 isolated at 16 weeks of gestation for all in vitro assays (ATCC® CCL-186™). pLNCX2 ER:rasG12V-expressing IMR90 were maintained and senescence induced as described under 5% O2 conditions. ER:IMR90 cells were co-cultured with IMR90:GFP (an empty vector fused with mVenus or with a dominant negative form of MAML1 fused with mVenus cells at 1:10 ratio. (Transwell bulk-RNA-seq): ER:RasG12V-expressing cells were co-cultured with IMR90:GFP. The co-cultured cells were placed into the lower chamber of a transwell system (density 5x103 cells/well). Another pure population of IMR90:GFP cells were cultured in the upper chamber of the transwell system. All cells were maintained in 4-hydroxytamoxifen (Sigma) for 7 days. All experiments were performed in triplicate.
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