ECM organization is altered in golgin mutant cultures. (A and C) Confocal maximum projection images of non-permeabilized WT, GMAP210 KO (A), and Golgin-160 KO (C) RPE1 cell cultures immunolabeled for extracellular collagen type I (green), fibronectin-1 (magenta), and nuclei (DAPI, blue). Scale bars, 10 µm. (B and D) Quantification of fibril characteristics measured from images represented in A and C using the TWOMBLI ImageJ plug-in (see Materials and methods). Individual dots represent the mean of each biological replicate (n = 4), and bars represent the median of all experiments. (E) Decellularized ECM from WT, GMAP210 KO, and Golgin-160 KO cell cultures imaged by HS-AFM. Images are representative of 10 × 10 raster scans from biological replicates (n = 3). Scale bars, 10 µm. (F) TWOMBLI quantification of fibril characteristics measured from images represented in E. Individual dots are measurements from each tile in the raster scan, with each biological replicate color-coded (n = 4). Bars represent the median of all experiments. (B, D, and F) Data were subjected to a Shapiro–Wilk test for normality and then a nested one-way ANOVA with Dunnett’s test for multiple comparisons to generate P values. ANOVA, analysis of variance.