Effects of Rtn4’s distribution on microscale ER structure. (A and C) Two-color live-cell STED images of U-2 OS Rtn4-Halo (green) CRISPR cells overexpressing SNAP-Sec61β (magenta). (A) Brown and yellow boxes are highlighting where regions of interest were made for Rtn4 pixel intensity measurements in peripheral and perinuclear tubules, respectively. (B) Histogram of average Rtn4 pixel intensity measured along tubules (P < 0.05; Wilcoxon rank-sum statistic). (C) Orange and gray boxes are highlighting where line plots were made for tubule diameter measurements using the NEP fitting method based on SNAP-Sec61β fluorescence for peripheral and perinuclear tubules, respectively. (D) Histogram of diameters of peripheral and perinuclear tubules (P < 0.01; Wilcoxon rank-sum statistic). (E and F) STED images of Halo-Sec61β overexpressed in U-2 OS and U-2 OS Rtn4-KO cells, respectively. Red and blue boxes represent where line plots were created for tubule diameter measurements for each dataset. (G) Diameters of U-2 OS and U-2 OS Rtn4-KO cells’ tubules (P < 0.001; Wilcoxon rank-sum statistic). (B, D, and G) Hashed vertical lines represent the average values that appear near each line. (A, C, E, and F) The scale bars represent 1 µm.