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To generate cells of different sizes, we made a doxycycline-inducible system that knocks down or overexpresses the G1 cyclin CCND1 in hTERT RPE-1 cells. This allowed us to control the activity of the CCND1–CDK4/6 complex and therefore, the pace of cell cycle progression. Since cell growth can be decoupled from the cell cycle, cells that spent longer in G1 grew larger, and cells that spent shorter amount of time in G1 grew smaller. This system enabled steady-state multi-omic profiling of differently-sized cycling cells.

To track in real-time how transcription is regulated by cell size, we used MS2 live-cell imaging. MS2 stem-loop sequences were incorporated into the endogenous introns of RAB7A and RHOA in near-diploid HBEC-kt3 cells. Binding of fluorescently-tagged MS2 coat protein to nascent pre-mRNA enables real-time visualization of individual transcriptional loci. A custom tracking algorithm extracted burst ON time, OFF time, amplitude, and size from fluorescence traces as a function of nuclear area — a proxy for cell size.

To ask how mRNA concentrations change with cell size, we performed RNAseq on CCND1-manipulated RPE1s of different sizes. Rapid processing immediately after induction avoided sorting-related mRNA degradation artifacts. Size-dependent transcript slopes were derived similarly to protein slopes to directly compare mRNA and protein concentration changes across a two-fold size range.

To track the rate of mRNA decay as a function of cell size, we used SLAMseq. Cells were pulsed with a uridine analog, 4-thiouridine (s4U), which is incorporated into newly transcribed RNA. A chemical conversion step marks s4U as a T-to-C substitution in sequencing reads. By tracking the decay of labeled transcripts during a chase period, individual mRNA half-lives were measured across different cell sizes.

To confirm that size-dependent proteome remodeling occurs in our genetically manipulated cells, we used isobaric tandem mass tag (TMT) labeling for multiplexed protein quantification. Protein size-slopes — the rate at which a protein's concentration changes with cell size — were derived and correlated with our previsouly published data from size-sorted immortalized cells, primary human cells, and primary mouse liver.

To track protein decay rates across cell sizes, we used stable isotope labeling by amino acids in cell culture (SILAC) combined with TMT multiplexing. Isotopically-labeled amino acids are incorporated into newly synthesized proteins and detected by mass spectrometry. By tracking the rate at which labeled proteins replace unlabeled ones, we estimated protein half-lives (T50) in small and large cells over a ten-day labeling period.

Lysosomal proteins are among the highest super-scaling proteins. To test whether this reflects a functional change in lysosome dependence with cell size, we challenged CCND1-manipulated cells with two lysosomal inhibitors: LLoMe (a lysosomotropic agent that physically ruptures the lysosomal membrane) at 3 mM for 3 hours, and chloroquine (which perturbs lysosomal pH and enzyme activity) at 50 µM for 48 hours. Cell death was quantified by Annexin V staining and cell permeability dye via flow cytometry. As a specificity control, cells were also treated with the proteasome inhibitor bortezomib (20 nM, 2 days).