FIGURE 4.

Deletion of Rb1 weakens G1/S cell size control in primary hepatocytes. (A) Schematic of the experiment used to examine the role of Rb1 in hepatocyte cell size control. (B) Live cell imaging traces of the Cdt1-mCherry and Geminin-Venus cell cycle reporters in Fucci2 hepatocytes grown in 2D culture. The G1/S transition is determined using the inflection point of the Geminin-Venus signal and the point of maximum Cdt1-mCherry signal. (C) Cell size and cell cycle statistics of Rb1 ^+/+ and Rb1 ^−/− hepatocytes. Cell size is approximated using nuclear area. (D) The correlations between nuclear area at birth and the nuclear area changes during G1 in Rb1 ^+/+ and Rb1 ^−/− hepatocytes. The shaded area indicates the 95% confidence interval. (E) The slope values of the linear relationship in (D) are plotted from three independent biological replicates. Each experiment used hepatocytes from different mice, and Rb1 ^+/+ and Rb1 ^−/− hepatocytes for each replicate experiment were derived from the same mouse. Dashed lines link experiments performed using hepatocytes derived from the same mouse. (F) p107 mRNA measured by RT-qPCR from Rb1 ^+/+ and Rb1 ^−/− 2D cultured primary hepatocytes (left panel) and Rosa-rtTA; TRE-shRb1 mice (right panel). The error bars indicate standard deviation. *p < 0.05, **p < 0.01. (G) Model indicating multiple mechanisms through which RB regulates cell size at the G1/S transition.