Figure 6.
Preferential elimination of large than normal-sized cells from proliferating populations alters cell size homeostasis
(A) Nuclear area of proliferating G1 cells as well as G1 and S phase cells after 3 h of palbociclib release. The vertical red bars show mean nuclear size. n = 140, 72, and 86 for each group. RPE1-mScarlet-PCNA cells were treated with or without 200 nM palbociclib for 48 h.
(B) Differential interference contrast (DIC) images, PCNA fluorescence (PCNA) as well as the nuclei of the three cells treated with palbociclib and ML162 cells as identified by the tracking software (SC-Track) at the indicated times. Scale bar: 10 μm.
(C) Single cell traces of nuclear area changes in control and 2-day palbociclib-treated cells immediately after ML162 addition. Red and blue lines show the average behavior of cells with larger or smaller than 200 μm2 nuclear size, respectively. The single large outlier cell in control sample was excluded from mean nuclear size calculations. n = 100 for control and 158 for palbociclib groups.
(D) Predicted and observed effect of RSL3 on cell size distribution in a proliferating cell population. Dashed black line overlaid with the observed cell sizes shows normal distribution. Note that swelling (see Figure 3B) shifts the size distribution slightly to the right.
(E) Skewness in RPE1 populations treated with varying concentrations of RSL3 (left) or combinations of RSL3 and Fer-1 (right).
(F) Skewness in MDA-MB-231 populations. For experiments with Fer-1, cells were cultured for 3 days with and without 1 μM Fer-1 and either 100 nM (for RPE1) or 500 nM (for MDA-MB-231) RSL3 was added for 24 h. Data shown are mean ± SD, n = 3. ANOVA followed by Tukey’s post-hoc test. See also Figure S6 and Videos S1, and S2.