Fig. 2: Arrayed screens validate genes involved in retrograde trafficking and transport to and from Golgi compartment.

a Schematic of arrayed validation screens. The sgRNA were delivered via lentiviral transduction. Following antibiotic selection, cells were treated with SS-ASO (25 nM, 3 days), and quantified the GFP+ expression by flow cytometry or high content imaging. Created in BioRender. Roudnicky, F. (2025) https://BioRender.com/vr5pkra. b Percentage of GFP+ cells was measured with flow cytometry. The screen was performed three times as independent experiments using 3 to 4 sgRNA per gene. Data is plotted as mean with SD and normalized to non-targeting sgRNA. c Proportion of GFP+ cells was measured using high content imaging. The screen was performed as 2 independent experiments using 3 to 4 sgRNA per gene. Data is plotted as mean with SD. d Representative images of the most interesting hits from the screen performed in Fig. 2c. Cellular segmentation was performed according to the Hoechst counterstaining and is depicted as white outlines. GFP is depicted in green. e Protein-protein association network generated by STRING database performed on the hits presented on (c). The disconnected nodes are not displayed. The thickness of the line between nodes represents the confidence of the interaction, with the thickest lines showing the highest confidence. The nodes are color-coded in red or green according to their effect on ASO activity. This analysis identifies the AP1 adapter complex, as well as the TBC1D123/Fam91a1/WDR1 complexes, outlining the importance of vesicular transport between endosome/Golgi in determining ASO activity. Source data are provided as a Source Data file or in corresponding Supplementary Data.