Fig. 2

LDs accumulate in the patient fibroblasts and can be reversed by overexpressing ABHD5-WT. (A) Images of representative cells detected in the CDS patient. Lipid vacuoles are marked by black arrowheads. (B) Primary skin fibroblasts derived from the proband and from a healthy individual (GM00498 cells) were stained with WGA for membrane segmentation (magenta), and with BODIPY to mark LDs (green). DNA was stained with Hoechst H33342 (blue). Enlargements of designated areas are in the boxed regions at right as numbered. Scale bars = 20 μm. (C-D) Western blot analysis of proteins extracts from skin fibroblasts derived from CDS patient cells, and from control skin fibroblasts cells (GM40098). Anti-Vinculin was used for loading control. Blots were incubated with (C) anti-ABHD5 and (D) anti-ATGL. (E) Quantifications of ATGL signal levels as seen in D. Data were analyzed using Fiji, and statistical analysis was carried out by two-tailed T-test (n = 3, ns = non-significant). Bar graph illustrates the mean ± SD. (F) Immortalized skin fibroblasts originating from the proband were electroporated with GFP-ABHDT-WT plasmid (green), followed by fixation after 24 h, and BODIPY staining to mark LDs (magenta). The experiment was performed three times (n = 12 positive cells). Enlargements of designated areas are in the boxed regions at right as numbered. Scale bar = 20 μm