High-throughput microscopy-based measurement of cell death susceptibility in different-sized cells.

(A) Experiment schematic: a genetic construct encoding nuclear-localized fluorescent protein mKate2 was delivered into HMEC cells using lentiviral transduction. After selection, these cells were FACS-sorted by cell cycle phase and cell size into small and large G1 cells (smallest and largest 5%). The FACS-sorted cells were seeded on 384-well plates and allowed to settle overnight before being treated with death-inducing compounds in the media containing a dead-cell dye SYTOX Green. The cells were imaged for 72 h during treatment, and live cells were identified by the presence of nuclear mKate2 fluorescence, while dead cells were identified by the presence of the SYTOX Green signal (Forcina et al, 2017; Inde et al, 2020). The numbers of live and dead cells were automatically tracked over time to determine the dose-response curves and cell death kinetics. (B) Cell size distributions of small and large G1-phase HMEC cells after FACS sorting, measured on a Coulter counter. (C) Dose-response curves of small and large FACS-sorted HMEC cells treated with a potent ferroptosis-inducing compound erastin2 (Era2). Rep1 and rep2 denote two different biological replicates, each with two technical replicates. (D) Cell death kinetics of small and large FACS-sorted HMEC cells treated with 10 µM Era2. Dots show the means of two biological replicates, error bars denote the range. (E) Dose-response curves of small and large FACS-sorted HMEC cells treated for 72 h with other lethal compounds: puromycin, doxorubicin, anisomycin, tunicamycin and bortezomib. Each experiment was performed twice independently, and each biological replicate included two technical replicates. The dots show averages of two biological replicates.

Larger cells are less susceptible to Era2-induced ferroptosis.

(A) Cumulative cell size distributions of HT-1080 cells, whose cell cycle was arrested in G1 phase for 0, 2, 3, 5, or 6 days with 1 µM of the CDK4/6 inhibitor palbociclib. (B) Cell survival percentage of HT-1080 cells whose size was increased through palbociclib-induced cell cycle arrest. After pre-treatment with palbociclib for the indicated numbers of days, the cells were exposed to 0.4 µM Era2 in the presence of palbociclib for 24 h. Cell survival percentage was calculated relative to the cells treated only with palbociclib. (C) Cell cycle phase distribution of asynchronously proliferating RPE-1 cells, and RPE-1 cells treated with the CDK4/6 inhibitor palbociclib for 2 or 4 days. (D) Cell size distribution of asynchronously proliferating RPE-1 cells, and RPE-1 cells treated with the CDK4/6 inhibitor palbociclib for 2 or 4 days. The numbers next to the histograms indicate mean cell size ± standard deviation for the corresponding condition. (E) Cell survival percentage in RPE-1 cells whose cell cycle was synchronized and size was increased through 2-day or 4-day palbociclib-induced cell cycle arrest. After pre-treatment with palbociclib for the indicated numbers of days, the cells were exposed to 0.4 µM Era2 in the presence of palbociclib for 20 h. Cell survival percentage was calculated relative to the cells treated only with palbociclib. (F) Cell size distributions of RPE-1 cells that inducibly express shRNA against cyclin D1 gene (CCND1) to slow down the cell cycle and increase cell size. Cells were grown in medium containing 500 ng/mL doxycycline for 4 days to induce shRNA expression and increase cell size. Uninduced cells were grown in the same medium with DMSO in place of doxycycline. (G) Cell survival percentage in control RPE-1 cells and cells expressing an shRNA against cyclin D1 gene (CCND1). Cells were treated with 0.2 µM Era2 for 48 h, and cell survival percentage was calculated relative to DMSO-treated cells. Cell survival percentages in graphs (B), (E) and (G) are shown as means ± s.e.m.; n = 3 biological replicates. A two-tailed unpaired Student’s t-test was used to evaluate the statistical significance of survival percentage differences in panels (E) and (G). (H) Graphical summary: Larger cells are less susceptible to Era2-induced ferroptosis.

Membrane lipid peroxidation decreases with cell size.

(A) Schematic of pathways regulating ferroptosis. Ferroptosis is induced through the accumulation of toxic peroxidized lipid species in the plasma membrane. The accumulation of peroxidized lipids is prevented by glutathione (GSH) production and GSH-dependent reduction of the peroxidized lipids (Jiang et al, 2021; Dixon & Olzmann, 2024). (B) The ratiometric fluorescent dye BODIPY-C11 581/591 detects lipid peroxidation in live HMEC cells (Dixon et al, 2012; Pap et al, 1999). Oxidation of the polyunsaturated butadienyl portion of this fatty acid analog results in a shift of the fluorescence emission peak from red (∼590 nm) to green (∼510 nm), allowing ratiometric analysis of lipid peroxidation using flow cytometry. Treatment of cells with Era2 increases lipid peroxidation, as indicated by decreased non-oxidized (red) BODIPY-C11 fluorescence and increased oxidized (green) BODIPY-C11 fluorescence. (C) A flow cytometry analysis of lipid peroxidation in different-sized RPE-1 cells within the DMSO-treated cell culture. The side-scatter parameter (SSC-A) was used as a proxy for cell size (Lanz et al, 2022; Tzur et al, 2011). Three biological replicates were performed, and 100,000 events were recorded for each. (D) A quantitative analysis of the flow cytometry data from panel (C): the data were binned into 12 bins by normalized cell size (SSC-A / median SSC-A), and the mean values for of oxidized (green) to non-oxidized (red) BODIPY-C11 ratio were plotted for each bin (black line). Gray shaded area denotes the s.e.m. for each bin, and the blue area denotes the standard deviation. (E) For Era2-treated cell populations, live cells were identified and gated in the indicated region using a live/dead cell permeability dye SYTOX Blue. (F) Size-dependent lipid peroxidation in RPE-1 cells treated with 0.2 µM Era2 for 20 h. The analysis was performed as in panel (D). (G-I) Proteomics-based analysis of GPX4 (G), ferritin heavy chain (H) and ferritin light chain (I) expression in FACS-sorted small, medium and large RPE-1 cells and primary lung fibroblasts (HLF). Each line in the plot corresponds to a unique peptide from the indicated proteins identified by mass spectrometry reported in Lanz et al. (Lanz et al, 2022).

Larger cells have higher concentrations of glutathione and enzymes promoting glutathione synthesis.

(A) Proteomics-based analysis indicates the concentrations of key enzymes involved in glutathione production (see Fig. 3A) increase with cell size. For this analysis, RPE-1 cells were FACS-sorted into populations of small, medium, and large G1 cells, and the proteomes of cells in these bins were analyzed using SILAC mass spectrometry. The primary proteomics data used to plot the concentrations of glutathione synthetase (GSS), glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits were taken from our previous work (Lanz et al, 2022). Each line in the plot corresponds to a unique peptide corresponding to the indicated protein that was identified by mass spectrometry. (B) Flow-cytometry-based measurement of cystine/glutamate transporter SLC7A11 (xCT) and cathepsin B (CatB) concentrations in G1-phase RPE-1 cells demonstrates a modest decrease in SLC7A11 and a significant increase in cathepsin B concentrations with cell size. To calculate the concentrations of SLC7A11 and CatB, their amounts were measured with flow cytometry using immunofluorescence and normalized to the amounts of α-Tubulin. The data were binned by cell size, and mean values for each bin were plotted against normalized cell size (solid blue line for SLC7A11 and red line for CatB). Shaded areas denote the s.e.m. for each bin. (C, D) Flow-cytometry-based measurement of GSH amount (C) and total protein amount (D) scaling with cell size in RPE-1 cells. The side scatter parameter (SSC-A) is used as a proxy for cell size (Tzur et al, 2011; Berenson et al, 2019). Three biological replicates were performed, and 100,000 events were recorded in each replicate. (E) GSH concentration in RPE-1 cells plotted against cell size. The GSH concentration is calculated as the ratio of GSH amount to the amount of total protein from data shown in panels (C) and (D). (F-H) Analysis of the flow cytometry data shown in panels (C-E). The data were binned by cell size, and mean values (black lines in the plots) for GSH amount (F), total protein amount (G) and GSH concentration (H) were plotted against normalized cell size (SSC-A / median SSC-A). Gray shaded areas denote the s.e.m. for each bin, and the blue area denotes the standard deviation. The orange line in (F) and (G) is shown for reference and corresponds to a perfect scaling scenario, where the amount of a cell component increases in direct proportion to cell size so that its concentration does not change. The total amount of protein is very close to perfect scaling, while the amount of GSH increases faster than cell size so that its concentration is higher in larger cells. (I, J) Comparison of GSH concentrations in small, medium, and large cells. Based on the flow cytometry data, 5% smallest, 5% largest and 5% intermediate-sized RPE-1 cells were gated (I), and their GSH concentration (GSH amount per total protein amount) histograms were plotted for each of these size populations (J). The plot shows that the GSH concentration distributions progressively shift towards higher values when cell size increases. The plots in (I-J) are based on the primary data shown in panels (C-D).

Higher expression of ACSL4 in smaller cells drives increased lipid peroxidation and ferroptosis.

(A) Proteomics analysis identified the size-dependent expression of ACSL4, an enzyme that enriches cellular membranes with long polyunsaturated fatty acids prone to peroxidation (Doll et al, 2017). The primary proteomics data were from our previous work (Lanz et al, 2022). Each line in the plot corresponds to a unique peptide from the ACSL4 protein identified by mass spectrometry; the dashed black line indicated the average ACSL4 protein slope. (B) Flow cytometry analysis confirms the decrease of ACSL4 concentration with cell size in HMEC cells. β-Actin was measured as a reference protein, as its concentration does not change with cell size. To calculate the concentrations of ACSL4 and β-Actin, their amounts were measured with flow cytometry using immunofluorescence and were normalized to the amounts of α-Tubulin. The data were binned by cell size, and mean values for each bin were plotted against normalized cell size (solid blue line for ACSL4 and black line for β-Actin). Shaded areas denote the s.e.m. for each bin. (C) Validation of ACSL4 knockout in HT-1080 cells with immunoblotting. Wild-type (WT) HT-1080 cell line and two different ACSL4 knockout clones (KO1 and KO2) were analyzed using antibodies against ACSL4 and α-Tubulin as a loading control. Bar plot shows the quantification of immunoblotting data from three biological replicates. (D) Deletion of ACSL4 eliminates the size-dependence of membrane lipid peroxidation in HT-1080 cells. The plot shows the flow cytometry measurements of lipid peroxidation after 16 h 1 µM Era2 treatment in wild-type and ACSL4 KO HT-1080 cells (ACSL4 KO1 and ACSL4 KO2 are two different knock-out clones) (Magtanong et al, 2019). The ratio between oxidized (green) BODIPY-C11 and non-oxidized (red) BODIPY-C11 fluorescence is plotted as a metric for lipid peroxidation, and the side-scatter parameter (SSC-A) is used as a proxy for cell size. The flow cytometry data were binned by cell size, and mean values of oxidized to non-oxidized BODIPY-C11 ratios were plotted for each bin (blue solid line corresponds to wild-type cells, orange and red lines correspond to ACSL4 gene-disrupted clones KO1 and KO2). Shaded areas denote the s.e.m. for each bin. (E) Cell size distributions of small, medium, and large G1-arrested ASCL4 KO HMEC cells isolated by FACS sorting. Prior to FACS sorting, the cells were cultured for 24 h in the presence of 1 µM palbociclib to synchronize cells in G1 phase. Cell size after sorting was measured on a Coulter counter. (F) Cell survival percentage in WT and ACSL4 KO HMEC cells, sorted into small, medium, and large size bins by FACS. After sorting, the cells were re-plated in the presence of 1 µM palbociclib to keep them in G1 phase. Cells were then treated with 10 µM Era2 for 52 h, and the cell survival percentage was calculated relative to palbociclib-only treated cells. Cell survival percentages in graphs are shown as means ± s.e.m. for n = 3 biological replicates.

Cell size modulates cell susceptibility to ferroptosis.

Larger cells are less prone to Era2-induced cell death because they generate less peroxidized plasma membrane lipids and more glutathione to reduce those toxic peroxidized lipids.

Effects of cell size on Era2- and RSL3-induced ferroptosis susceptibility in HT-1080 cells.

(A) Cell survival percentage in HT-1080 cells sorted into four cell size bins using FACS. Cells were treated with 0.3 µM Era2 for 48 h, and cell survival percentage was calculated relative to DMSO-treated cells. The plot shows a representative example from n = 2 biological replicates. Each biological replicate included two technical replicates per condition, which were averaged for quantification of cell size and cell death sensitivity. (B) Cell survival percentage in HT-1080 cells whose size was increased through palbociclib-induced cell cycle arrest. After pre-treatment with palbociclib for the indicated numbers of days, the cells were exposed to 0.7 µM RSL3 in the presence of palbociclib for 24 h. Cell survival percentage was calculated relative to the cells treated only with palbociclib. Cell survival percentages are shown as means ± s.e.m.; n = 3 biological replicates.

Cell size does not affect the concentrations of labile and lysosome-sequestered iron in RPE-1 cells.

(A,B) Flow-cytometry-based measurements of the amount of intracellular labile iron (A) and lysosome-sequestered iron (B) in G1-phase RPE-1 cells. Intracellular labile iron was detected with the FerroOrange fluorescent probe, and lysosomal iron was detected with the Lyso-FerroRed probe. Total cellular protein amount (CFSE staining), which scales in proportion to cell volume, is shown for reference. G1 cells were gated based on Hoechst DNA staining. The data were binned by cell size, and mean values for each bin were plotted against normalized cell size. Shaded areas denote the s.e.m. for each bin. Each sample included 100,000 cells.

Senescent cells are large and resistant to Era2-induced ferroptosis.

(A) Cell size distributions of senescent and non-senescent (asynchronously proliferating) RPE-1 cells, measured with a Coulter counter. The numbers next to the histograms indicate mean cell size ± standard deviation for each condition. 4-day treatment with 100 nM doxorubicin was used to induce senescence in RPE-1 cells. To reduce the size of senescent cells, 100 nM of the mTORC1 inhibitor rapamycin was added to the media during the 4-day doxorubicin treatment. (B) Cell survival percentage in senescent and non-senescent RPE-1 cells treated with 0.4 µM Era2 for 23 h. Senescent cells demonstrate significantly higher resistance to Era2 than non-senescent cells. Senescent cells pre-treated with rapamycin show only a modest reduction in cell survival. Cell survival percentage was calculated relative to the cells under the same conditions treated with DMSO instead of Era2. Error bars show s.e.m., n = 3 biological replicates. (C) Prolonged cell pre-treatment with 100 nM mTORC1 inhibitor rapamycin causes a moderate decrease in cell sensitivity to Era2.

FACS-sorted ACSL4 KO HMEC cells have size distributions similar to those of WT HMEC cells.

(A) Validation of ACSL4 knockout in HMEC cells with immunoblotting. Wild-type (WT) cell line and a mixed (not clonal) population of ACSL4 knockout cells were analyzed using antibodies against ACSL4. (B) Mean cell sizes of small, medium, and large WT and ACSL KO HMEC cells after FACS sorting, measured on a Coulter counter. Error bars indicate cell size S.D. Prior to the sorting, the cells were synchronized in G1 phase by a 24 h treatment with 1 µM palbociclib.

Flow cytometry gating strategy for selecting G1 cells for subsequent scaling analysis.

Single cells were gated based on FSC-A vs SSC-A, then FSC-A vs FSC-H, then SSC-A vs SSC-W plots. From this population of single cells, G1 cells were selected using Hoechst-A vs FSC-A plot for subsequent scaling analysis (the example shown is for cathepsin B scaling analysis).