(A) The apparent elastic modulus of S. cerevisiae spheroplasts (without rigid cell walls) at pH 7.4 (E = 636 ± 16 Pa (mean ± SEM); N = 249) and pH 6.0 (E = 1459 ± 59 Pa; N = 257) was measured by AFM-based indentation. The cytosolic pH of spheroplasts was adjusted with phosphate buffers of pH 6.0 and pH 7.4, respectively, containing 2 mM DNP, 1% glucose and 1 M sorbitol. (B) The same cells as in (A) characterized with real-time deformability cytometry (RT-DC). Each measured cell results in a dot in this deformation-cell diameter plot. Also shown are 90% (solid) and 50% (dashed) density lines, and the histograms of size and deformation including Gaussian fits. (C) The cell wall of rod-shaped S. pombe cells was removed under control, energy depletion, and pH-adjusted conditions. The cytosolic pH of cells was adjusted during spheroplasting with phosphate buffers of pH 5.5 and pH 7.4, respectively, containing 2 mM DNP, 2% glucose, 1 M sorbitol and cell wall-digesting enzymes. Cells were energy-depleted in growth medium without glucose containing 20 mM 2-deoxyglucose and 10 µM antimycin A for 2 hr prior to spheroplasting. Energy depletion was continued during spheroplasting by including 20 mM 2-deoxyglucose and 10 µM antimycin A in the spheroplasting buffer. (D) The roundness of more than 160 cells per condition at the start of the experiment and after 3 hr of incubation in the presence of cell wall digesting enzymes (end) was quantified. ∗∗p<0.01; ∗∗∗p<0.001.