Figure 8. Enlarged cells are more resistant to additional stress.

(A) Scheme of cell size enlargement or reduction experiments. CHX, cycloheximide; CDKi, CDK4/6 inhibitor. (B) Cell size was first enlarged by treating cells with 100 nM CDKi for 3 days; then, cells were washed and subjected to chronic HS at 40 °C for 3 more days (CDKi >HS). Cell size (% FSC-MFI; grey part of the bars) and cell death (% annexin V and PI-positive; red part of the bars) were measured by flow cytometry. The values for cell size and death in the different experimental conditions are normalized to the respective 37 °C controls (n=3 biologically independent experiments). (C) Cells were first pretreated with 7.5 nM rapamycin (Rapa) for 3 days to reduce the cell size. After that, the cells were subjected to chronic HS at 40 °C for 3 days (Rapa >HS). HS >Rapa, the two treatments were done the other way around. The cell size (% FSC-MFI) and relative cell death (% annexin V and PI-positive) were quantified by flow cytometry. The values for cell size and death in different experimental conditions are normalized to the respective 37 °C control (n=3 biologically independent experiments). (D) Scheme of experiments aimed at determining impact of limiting physical space on cell size increase. (E and F) Phase-contrast micrographs of RPE1 cells seeded in different numbers to restrict the space for cell size increase during adaptation to chronic HS (representative images of n=4 biologically independent experiments). The size bar in the top left panel indicates 100 µM. The cell size (% FSC-MFI) and relative cell death (% annexin V-PI positive) are quantified by flow cytometry. For the bar graphs, the values for cell size and death in different conditions are normalized to the low density (dns) cell population at 37 °C day 0 (n=4 biologically independent experiments). The data are represented as mean values ± SEM for all bar graphs. The statistical significance between the groups was analyzed by two-tailed unpaired Student’s t-tests.

Figure 8—figure supplement 1. Smaller cells are more susceptible to additional stress.

(A) Cell size was enlarged by treating cells with 100 nM CDKi for 3 days. Fold change of cell size (represented by the FSC-MFI values) and total proteins (determined as MFI-FL1 values) were analyzed by flow cytometry. Cells were fixed, and total proteins were stained using Alexa Fluor 488 NHS ester (n=3 biologically independent experiments). (B) Coomassie-stained protein gel showing protein samples from equal numbers of cells treated or not with CDKi (representative images of n=2 biologically independent experiments). (C) Cell size was enlarged by treating cells with CDKi for 3 days, before cells were treated with sodium arsenite (40 μM) for 3 days. Cell size (% MFI) and cell death (% annexin V and PI-positive) are measured by flow cytometry. α KO, Hsp90α KO; β KO, Hsp90β KO cells. (D) Order of treatment experiment as in Figure 8C, but with HEK cells. Cell size (% MFI) and relative cell death (% annexin V and PI-positive) are quantified by flow cytometry. The values for cell size and death in the different experimental conditions are normalized to the respective 37 °C controls (n=3 biologically independent experiments). (E) Cell size was reduced by serum starvation (starved) for 3 days before subjecting cells to chronic HS for 3 additional days (starved >HS). HS >starved, the two treatments were done the other way around (HS >starved). Cell size (% MFI) and relative cell death (% annexin V-PI positive) were quantified by flow cytometry and normalized to the respective 37 °C controls (n=3 experimental samples). (F) Order of treatment experiment with CHX and rapamycin to reduce cell size first for 3 days and then subjecting cells to oxidative stress with 10 µM sodium arsenite (Ars) for 1 day. Cell size (% MFI) and relative cell death (% annexin V and PI-positive) were quantified by flow cytometry and normalized to the Ars single treatment controls (n=3 experimental samples).