Time course for the uptake of plant PeptoQ into tobacco BY-2 cells. Cells were treated at day 3 after subcultivation (at the completion of the proliferation phase of the culture) with 2 µM of plant PeptoQ and followed by spinning disc confocal microscopy making use of the fluorescent signal of the conjugated rhodamine. The peptoid was present throughout the experiment. Geometric projections of confocal z-stacks collected from representative cells recorded at constant laser power and exposure time are shown in (A), The white squares mark the region of interest magnified in (B) to show the subcellular details. White arrows indicate punctate structures that are seen in early time points which represent endoplasmic reticulum (ER) structures that have active role in the internalization process, white arrowheads indicate filamentous structures. (C) Quantification of intracellular accumulation based on the integrated intensity inside the cell corrected for background either under continuous presence of 2 µM plant PeptoQ (white squares, solid curve) or as pulse-chase, where cells were washed after one hour of incubation with 2 µM and then followed further (grey squares, dashed curve). Mean values and standard errors from three independent experiments are shown. (D) Uptake determined from the first derivative of the cumulative time course shown in (C) to show the non-steady nature of the process, where a first period of rapid uptake in the first 20 min is followed by a static period, and then the second wave of uptake became evident after 90 min. Beyond 120 min (at 180, 240, 300 and 360 min), uptake became saturated.