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Expression vectors used in this study all contained the same CMV promoter and essentially the same plasmid backbone (p21-pRc/CMV, a gift from William Kaelin via Addgene; pCDNA3-pRb, a gift from Joseph Nevins; pCMV-p130, from OriGene; pCDNA3-FLAG-Myc and pCDNA3-FLAG-CycD1, from GenScript; and pCMV-mCherry, a gift from Lingchong You). Cells were then incubated at RT for 2 hr with protein-specific primary antibodies (p21(c-19), sc-397, Santa Cruz Biotechnology; p130(c-20), sc-317, Santa Cruz Biotechnology; pRb(IF8), sc-102FITC, Santa Cruz Biotechnology; and for FLAG-Myc and FLAG-CycD1, DYKDDDDK Tag antibody [fluorescein isothiocyanate, FITC], A01632, GenScript), and subsequently stained at RT for 2 hr with an FITC-conjugated secondary antibody (sc-2012, Santa Cruz Biotechnology) when necessary and applicable. Antibody-stained cells were measured using an LSR II flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (v.10.0). |
Quiescence is a non-proliferative cellular state that is critical to tissue repair and regeneration. Although often described as the G0 phase, quiescence is not a single homogeneous state. As cells remain quiescent for longer durations, they move progressively deeper and display a reduced sensitivity to growth signals. Deep quiescent cells, unlike senescent cells, can still re-enter the cell cycle under physiological conditions. Mechanisms controlling quiescence depth are poorly understood, representing a currently underappreciated layer of complexity in growth control. Here, we show that the activation threshold of a Retinoblastoma (Rb)-E2F network switch controls quiescence depth. Particularly�... More