Figure 2
From: Disease relevance of rare VPS13B missense variants for neurodevelopmental Cohen syndrome
Golgi enrichment is affected by VPS13B missense variants. (A–D) Golgi fragmentation is rescued by wild type VPS13B overexpression. HeLa cells were transfected with either control (scrambled siRNA) (A,B) or a VPS13B-specific RNAi (siRNA #25721, ambion) (C,D). 48 h post RNAi-transfection HeLa cells were processed for overexpression and either nontransfected (A,C) or transfected with the pcDNA3.1_VPS13B-wt construct (B,D). 24 h later, all cells were processed for immunostaining of VPS13B (green) and GM130 (red). Imaging occurred with confocal microscopy. Nuclei were stained using DAPI, scale bars 10 µm. (E,F) Analysis of subcellular localization of cloned VPS13B missense variants. ImageJ analysis was used to evaluate Golgi localization of VPS13B missense variants. Two ROI (yellow) were defined for each cell: “whole cell” ROI was drawn at the outline of the cell to detect total immuofluorescence levels; “Golgi region” was defined by the counterstaining of GM130 (red). Fluorescence intensity of VPS13B (green) in each ROI was measured (E). Percentage of VPS13B enrichment at the Golgi was finally calculated for each missense variant using GraphPad prism. We repeated each condition at least 3 times and analyzed each time 3 recordings (with at least 1 transfected cell). Please refer to Supplementary table T3 for statistical information (F).
