Figure 8. Perturbing the relationship between cell cycle length and growth rate.

(A) Rpe1 cells were treated with varying doses of palbociclib (cdk4/6 inhibitor) or radicicol (hsp90 inhibitor). (A) The fold change in mean cell cycle length (left panel), growth rate (middle panel), and cell size (right panel), relative to untreated cells, is plotted for each condition. The mean cell cycle length and mean growth rate of cells in each condition were calculated from measured increases in bulk protein and number of cells over the course of a 68 hr incubation, as described in the Materials and methods and Results sections. Mean cell size (protein content assayed by SE-A647 staining) was measured after 68 hr of incubation in each condition. Each drug treatment was done in duplicate, alongside six control (DMSO) samples, with several thousand cells in each sample. Fold-changes are shown relative to the average cell cycle length, growth rate, and cell size of all six control samples. (B) Representative widefield fluorescence images (SE-A647 stain) of unperturbed cells and cells treated with 600 nM cycloheximide, 175 nM PHA848125, 5 uM Cdk2 Inhibitor III, 500 nM palbociclib, or 500 nM radicicol. All images are shown at the same scale, scale bar = 50 um. Red lines delineate cell boundaries. The raw data and source code necessary to generate (A) are included in Figure 8—source data 1.

Figure 8—figure supplement 1. siRNA knockdown of CDK4 and CDK6 causes an increase in cell size.

Rpe1 cells were transfected with 25 nM control siRNA (siCON) or 25 nM siRNA targeting: (1) CDK6, (2) CDK4, (3) both CDK4 and CDK6. (A,B) Cells were harvested at 48 and 72 hr post-transfection, for western blot verification of siRNA knockdown. Western blots probed for CDK4 (A) and CDK6 (B) confirm that these proteins are depleted after treatment with siCDK4 and siCDK6, respectively, but are present in control samples (siCON). (C) After 72 hr of siRNA treatment, cells were fixed and protein was stained with SE-A647 to measure cell size. Experiment was performed on five replicates for each condition (ten replicates for control), with several thousand cells in each replicate. Median cell size (protein content as measured by SE-A647 fluorescence) is shown for each condition, with error bars indicating the standard deviation of replicates.