FIGURE 5:

Amish GMS fibroblasts exhibit defects in autophagosomal biogenesis due to a loss in WHAMM function. (A) Fibroblasts from two healthy (+/+) Amish individuals or Amish GMS (GMS/GMS) patients were grown in the absence or presence of chloroquine (CQ) for 5 h and stained with antibodies to detect LC3A (red) and with DAPI to label DNA (blue). (B) The number of LC3A puncta per cell, and the relative area within each cell occupied by LC3⁺ structures was quantified using ImageJ. Each bar represents the mean (+ SEM) from analyses of 12 cells. ***, p < 0.001 (ANOVAs). (C) The percentage of cells with large cytoplasmic vacuoles was quantified. Each bar represents the mean (+ SEM) from analyses of 250 cells. ***, p < 0.001 (Fisher’s exact test). (D) Fibroblasts from a healthy individual or GMS patient were grown in the presence of rapamycin (RM) for 16 h and stained with antibodies to detect LC3B (red) and with DAPI to label DNA (blue). (E) Fibroblasts were treated with rapamycin for 16 h or starved for 90 min and stained as in D. The relative area within each cell occupied by LC3⁺ structures was quantified as in B. ***, p < 0.001 (t test). (F) Fibroblasts from a GMS patient were transfected with plasmids encoding GFP-WHAMM or GFP-WDR73, grown in the absence or presence of CQ, and stained as in A. Normal LC3 staining was observed in 29/35 GFP-WHAMM-expressing cells and 5/37 GFP-WDR73-expressing cells. All scale bars: 10 μm.