Fig. 7

Vacuolin-mediated inhibition of exocytosis normalizes cathepsin activity and restored TGFß signaling in neu1 and gnptab deficient larvae (A) In gel analyses of embryos labeled with the BMV109 activity-based probe show vacuolin treatment primarily reduces Ctsk activity in neu1 deficient animals and Ctsl in gnptab deficient animals. n = 3 independent experiments with 12 embryos per condition. (B) Graph shows quantitation of Ctsk and Ctsl activities in neu1 deficient animals. Error = SEM, significance was assessed using the Dunnet’s test (red stars), where ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (C) Graph shows quantitation of Ctsk and Ctsl activities in gnptab deficient embryos. Error = SEM, significance was assessed using the Dunnet’s test (red stars), where ∗∗∗p < 0.001. (D) Confocal analyses of pSmad2 staining in fli1a:EGFP positive neu1 and gnptab deficient cartilages treated with either DMSO or 35 nM vacuolin. Boxed areas shown as red only in panels to the right. For reference chondrocytes are outlined with dashed white lines. Percent values represent number of embryos exhibiting the pictured phenotype. n = 15 embryos per condition. (E and F) Graphs quantitating the percent of chondrocytes in neu1 or gnptab deficient larvae with nuclear localized pSmad2. Error = SEM, significance was assessed using the Dunnet’s test (red stars), where ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. See also Figure S4.