Panel A shows fluorescence microscopy images of PAUF-mRFP in Dimethyl sulfoxide and Tubacin treated cells, with dashed outlines indicating cell boundaries. Panel B shows a superplot of Number of CARTS per cell, with the x-axis labeled Dimethyl sulfoxide and Tubacin, and the y-axis labeled Number of CARTS per cell. Panel C shows a superplot of Cell adhesion area (square micrometers), with the x-axis labeled Dimethyl sulfoxide and Tubacin, and the y-axis labeled Cell adhesion area (square micrometers). Panel D shows a superplot of Number of CARTS per cell adhesion area (per square micrometer), with the x-axis labeled Dimethyl sulfoxide and Tubacin, and the y-axis labeled Number of CARTS per cell adhesion area (per square micrometer). Panel E shows fluorescence lifetime imaging of Halo-Flipper and ManII-Halo (JF646) in Control and Tubacin treated cells, with zoomed regions and a Halo-Flipper lifetime (nanoseconds) color scale. Panel F shows a superplot of Halo-Flipper lifetime (nanoseconds), with the x-axis labeled Dimethyl sulfoxide and Tubacin, and the y-axis labeled Halo-Flipper lifetime (nanoseconds).
MT hyperacetylation promotes CARTS biogenesis. (A) Confocal microscopy images of fixed HeLa cells transfected with the PAUF-RFP plasmid, subjected to a treatment with or without tubacin. Cell contours are indicated by dashed white curves. Scale bars, 10 µm. (B–D) SuperPlots showing individual cell measurements (small, light-colored symbols; N∼10 per biological replicate) and the mean value for each independent biological replicate (larger, black-outlined circles; n = 3) of B number of CARTS per cell, C cell adhesion area, and D number of CARTS per cell adhesion area. (E) FLIM Images of live RPE1-ManII-Halo stably expressing cells before and after overnight treatment with 10 µM of tubacin. The FLIM signal (Halo-Flipper lifetime) is displayed. Golgi was post-labeled using JF-646 (intensity shown, JF646 channel). Higher magnification of the boxed areas is shown on the right. Scale bar, 20 µm. (F) FLIM-measured Halo-Flipper fluorescence lifetime (in ns) was quantified for each experimental condition (n = 3 replicates; N∼10 cells per replicate). Two-sided parametric ratio paired t tests were used in all plots, and P values are indicated in the plots.