Fig. 2: Investigating the pH of the nucleolus and nucleoplasm under different physiological conditions.

a A schematic illustrating the relationship between nucleolar pH, POL1 (RNA polymerase I) activity, DNA replication, and the phase separation status of the nucleolus. b Representative confocal fluorescence images of different cells after treatment with either DMSO or CX-5461, incubated with SNARF-4F. c-j The 580/640 nm emission signal profile for the nucleolus and nucleoplasm in 3T3, OC1, HELA, and AML12 cells treated with (c–f) DMSO or (g–j) CX-5461. The white lines in (b) denote the region where the data points are taken. The light blue area in c-j highlights the location of the nucleolus. Violin plots showing nucleolar pH quantification in (k) 3T3, (l) OC1, (m) HELA, and (n) AML12 cells treated with DMSO or CX-5461. Dots represent the average pH values of both the nucleolus and the nucleoplasm. n = 30 independent biological replicates. Data are presented as mean ± standard deviation (SD). Statistical significance is determined using a two-sided Student’s t-test. o Confocal fluorescence images of HELA cells stained with SNARF-4F after treatment with different concentrations of Act D. p Profile of the 580/640 nm emission signal for the nucleus in HELA cells treated with different concentrations of Act D. The white lines in (o) denote the region where the data points are taken.