Figure 6. Inhibition of protein synthesis is accompanied by a similar decrease in nucleo-cytoplasmic crowding and does not perturb the N/C ratio.

(A) Overlay of the plasma membrane (green) and nuclear membrane (purple) of whole cells middle plane over time treated with 50 mg/ml cycloheximide (CHX, top) or not (Ctrl, bottom). (B) Single whole-cell volume dynamics treated with CHX or not (Ctrl). (C) Same as (B) for single-nucleus volume dynamics treated with CHX or not (Ctrl). (D) Individual whole cells N/C ratio dynamics treated with CHX or not (Ctrl). (E) Single whole-cell N/C ratio dynamics extracted from (D) for each condition. (F) Relative cytoplasmic (green) and nucleoplasmic (purple) GEM effective diffusion dynamics for cells treated with CHX (left panel) or only the drug buffer for control (0.5% dimethyl sulfoxide (DMSO), right panel). Statistical differences compared with Mann-Whitney U test. (G) Cells were stained for total protein and RNA using FITC dye. Left, confocal middle plane image of cells before (0 min) or after CHX treatment for 60 min. Right, quantification ratios of FITC intensities in nuclear and cytoplasmic regions over time after the addition of CHX. Kruskal-Wallis statistical test was used, from at two biological replicates. (H) Cytoplasmic (green, left) and nucleoplasmic (purple, right) protein signals for the same cells over time under CHX treatment decrease similarly. Kruskal-Wallis statistical test was used. Scale bar = 5 µm. See also Figure 6—figure supplement 1. (A-F &H) From at least three biological replicates.

Figure 6—figure supplement 1. CHX treatment produces no detectable change in N/C ratio.

(A) Bright field (top) and max Z-projection (bottom) overlay of the plasma membrane (green) and nuclear membrane (purple) of whole cells treated with 50 mg/ml CHX over time. Cells were selected to have approximately the same size. (B–C) Whole-cell volume and nuclear volume of distinct populations of cells treated with CHX and initial condition. (D) N/C ratio of a population of cells treated with CHX (light blue) and initial condition (blue). N≥253 per time point. (E) Cells were stained with FITC dye to quantify protein and RNA distribution along the long cell axis of untreated cells, normalized by cell length. (F) As in (E) except cells were treated with RNAse for staining for total protein. (E–F) Purple and green boxes indicate the positions used to measure nuclear and cytoplasmic signals in Figure 5G–H. Only a subpopulation of cells was plotted. (G) Confocal middle plane image of cells stained with FITC for total protein, before (0 min) or after CHX treatment for 60 min. Scale bar = 5 µm.