Changes in available GTP positively correlate with rates of NCT; however, this correlation was not observed in individual cells at steady state. (A) Correlation between the rate of NCT and available GTP with all treatments used in this study that significantly changed transport. All treatments are from BJ-5ta cells unless noted. (B) Plot of import rate and available GTP in BJ-5ta cells stably coexpressing LINuS and GEVAL30 at steady state. Results are from four independent experiments (n = 128). Pearson’s correlation is annotated for both A and B. (C) Measurements of NCT and GTP availability in individual BJ-5ta cells coexpressing LINuS and GEVAL30 before treatment (blue) and 10 min after cytochalasin B treatment (orange). Squares indicate the average GEVAL30 and import rate for each condition, with large unfilled circles highlighting the general distribution of cells within each treatment. Inset shows average NCT rate and GEVAL30 signal before and after cytochalasin B treatment in C. Results are from two independent experiments (n = 52). Significance calculated using a paired t test. P***<0.001, P****<0.0001. Error bars represent ± SEM. (D) Data from C was analyzed by normalizing the starting point for each cell to 0, with the length and direction of the lines indicating the extent of change. Colors of individual cell trajectories are defined in E. The percent of cells within each quadrant is indicated. (E) Cells in C prior to cytochalasin treatment were binned based on their GEVAL30 values and import rates to indicate cells that, compared to those within the 75% quartile (black), exhibited elevated levels of GEVAL30 (green), import rate (red), or both (orange). The listed percentage in each quadrant indicates the percent of cells within that quadrant that exhibited an increase in both GEVAL30 and import rate following cytochalasin b treatment. The measurements of those cells are depicted in the top right quadrant in D.