B cells activated on stiff substrates display enhanced tubulin acetylation and central clustering of lysosomes at the IS. (A) Representative images of fixed cells activated on soft or stiff substrates at different time points stained for α-Tub (red) and Ac-Tub (green). (B) Ac-Tub density calculations based on MFI at the IS for images in A. (C) Ac-Tub/α-Tub MFI ratios quantified at the IS for images in A. (D) Representative images of B cells seeded over stiff or soft substrates at different activation times. Ac-Tub is shown in green, LAMP1 in red, and the cell border (white dashed outline) was defined by F-actin phalloidin staining. (E) Quantification of lysosome accumulation on acetylated MT tracks from cells shown in D using Manders overlap coefficient. (F) Representative time-lapse images showing the tracking of cathepsin D⁺ lysosomes from cells activated on either stiff or soft conditions control (non-treated—NT) cells and SAHA-treated cells. Lysosome tracks were followed for 5 min after seeding the cells for 15 min. (G and H) Displacement and mean speed of lysosome tracks for experiments detailed in F. Data shown consider n ≥ 30 cells pooled from N = 3 independent experiments. Scale bars are 10 μm. Inset scale bars are 2 μm. Two-way ANOVA with Sidak’s multiple comparison test. P values illustrated with asterisks are * <0.05, ** <0.01, and **** <0.0001. Error bars are mean ± SEM.