Notably, actinomycin D had the same effect as cisplatin in HCT116 cells but not in U2OS cells, in which it rather caused a reduction in FBL and an increase in NPM1 phase separation, similar to oxaliplatin. Third, cisplatin showed a distinct thermodynamic profile compared with oxaliplatin, as it reduced ΔΔG tr of both FBL and NPM1. Notably, this mimics the greater overall sensitivity of HCT116 cells to oxaliplatin compared with U2OS cells ( Figure S2), suggesting that altered nucleolar phase separation drives the effects of oxaliplatin on cell viability. HCT116 cells were more sensitive for this reversal, as it occurred at oxaliplatin doses greater than 12.5 μM as opposed to 25 μM for U2OS cells. Second, with increasing doses of oxaliplatin, ΔΔG tr for NPM1 became less negative, demonstrating a reversal in the driving force for NPM1 phase separation. This corresponds to a reduced thermodynamic driving force for FBL phase separation and an increased one for NPM1, in agreement with the observed changes in nucleolar morphology. First, oxaliplatin oppositely affects ΔΔG tr of FBL and NPM1 in both HCT116 and U2OS cells, causing it to be positive for FBL and negative for NPM1.
This allowed us to construct thermodynamic maps for the direct comparison of drug effects on FBL and NPM1 phase separation, revealing three insights ( Figure 1H). Using ΔG tr in untreated cells as a reference, we then determined the changes in ΔG tr induced by a range of concentrations of oxaliplatin, cisplatin, and actinomycin D in both HCT116 and U2OS cells. ΔG tr is a function of the “width” of the two-phase regime (i.e., the ratio of the dense-phase concentration over the light-phase concentration Figure S3).
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