Supplemental data on imaging readouts for downstream analysis of screen hits. (A) Confirmation that dynein promotes perinuclear enrichment of Golgi and lysosomal membranes in U-2 OS cells. Shown are representative images and quantification of dispersion of trans-Golgi network (TGN46) and lysosomal membranes (LAMP1) in U-2 OS cells following the indicated treatments (Noc, nocodazole). Bar graphs show the ratio of spot number in the perinuclear region versus the peripheral region (lower values indicate increased dispersion). Data points represent mean per cell intensity values aggregated at the well level (minimum of 100 cells analyzed per well; four wells analyzed per condition). Error bars signify SD. ***P < 0.001 (one-way ANOVA with Dunnett’s multiple comparison versus NTC + DMSO). Scale bar, 20 µm. (B) Reproducibility of secondary screen biological replicates. Linear regression was performed on the main endpoints used in the two independent runs in the secondary screen. Data points represent the median rZ (central reference = NTC) of individual crRNA pools. The “proportion of cells with two γ-Tubulin puncta” had an R² score of <0.2 (0.07254) and therefore was not used for hit calling.