Fig. 1: Stx2 deletion increases exocytosis of predocked and newcomer granules in human β-cells.

a Representative western blot (n = 3 independent experiments, analysis in Supplementary Fig. 1a) showing the efficiency of Stx2 KD (see Methods) in human cells (HEK293A cell, human islets) and rat β-cells (INS832/13). β-actin used as loading control. b Representative TIRFM images monitoring the behavior of granules with the fluorescent granule marker neuropeptide-Y-EGFP (NPY-EGFP). Yellow circles indicate the sites of granule fusion events for Stx2-KD and Control (Ctrl) human β-cells. Granule trajectories are shown on the right with colors code of the times elapsed. These images are representative of n = 10 cells for Control and Stx2-KD from three independent experiments, from which the number of fusion events (c–f) and analysis of granule mobility (g, h) were assessed. Scale bar, 1 μm. The number of fusion events of predocked and newcomer in control (c) and Stx2 KD (d) human β-cells at 1-min intervals during first and second phases of glucose (16.8 mM) stimulated insulin secretion. Data obtained from 5 pancreatic islets donors and shown mean + s.e.m. Summary of fusion events of predocked (e) and newcomer (f) granules calculated from (c, d), showing Stx2-KD increased the exocytosis of predocked and newcomer granules. Data shown mean ± s.d., two-tailed unpaired Student’s t-test. Histogram of the granule diffusion coefficient D measured from the trajectory of individual granule in WT (g) and Stx2 knockdown (h) human β cell after glucose stimulation. Diffusion coefficient D is a measure of how constrained diffusion of the particles, smaller values indicate more restricted movement⁵². Dots represent raw data and the histogram was fitted with a three-component Gaussian function (defining 3 subpopulations indicated as 1, 2 and 3 in b and explained in the Results Section), sum of each peak was represented by black lines. The asterisk in h indicates that Stx2 KD β-cells had more granules that exhibited more restrictive movement (25.2% vs. 18.2% in Control β-cells) whereas the asterisk in g indicates that Control β-cells had more granules exhibiting less restrictive movement (23.9% vs. 14.1% in Stx2-KD β-cells). Source data are provided as a Source Data file.