Nuclear transport rates are dependent on the O-GlcNAc level. (A) Nuclear import (top) and export (bottom) rates after 3 d of transfection with scrambled (NegCtrl), OGT, or OGA siRNAs. n > 360 nuclei per condition. (B) Import (top) and export (bottom) rates vs. time after treatment with DMSO (black, solid), OSMI-4 (red, dashed), and Thiamet-G (blue, dotted) at 2.5 µM (left), 5 µM (middle), and 10 µM (right). Line and shaded area indicate mean and 95% confidence interval, respectively. n ≥ 120 nuclei per time point per condition. (C) Representative anti-O-GlcNAc (RL2) antibody immunofluorescence images of cells transfected with NegCtrl, OGT, or OGA siRNA for 3 d or treated with DMSO, 10 µM OSMI-4, or 10 µM Thiamet-G for 20 h. Scale bar, 50 µm. (D) Quantification of nuclear RL2 intensity (background corrected and normalized such that the median value of control condition is 1). n > 1,200 nuclei per condition. (E) Median import (left) or export (right) rate (from B) vs. median nuclear RL2 intensity (from D), measured after the knockdowns (circles) or inhibitions (triangles) of OGT (red) or OGA (black). Black line and gray shade are the line fit and its 95% confidence interval. P values were calculated by two-sided Welch’s t test for comparison with negative control. n.s., P > 0.01; *, P < 1 × 10−2; ***, P < 1 × 10−4.
