Figure S4.

StableMARK at low levels has minimal effects on MTs and organelle transport. Related to Figs. 4 and 5. (A–C) Independent replicates of the experiments shown in Fig. 4, A–D. (A) Fluorescence images of U2OS cells stained for acetylated tubulin and α-tubulin at different levels of StableMARK expression as analyzed in B. (B) Quantification showing the intensity ratio of acetylated tubulin (I_A) over total tubulin (I_T), plotted against the intensity ratio of StableMARK (I_SM) over total tubulin for individual StableMARK-expressing cells (each dot represents a single cell, n = 131, N = 1; see Fig. 4 for replicate). Solid green line + dashed lines indicate mean ± SD of the intensity ratio of acetylated tubulin over total tubulin for non-expressing cells (n = 120). Green data points represent low expression example from A. Orange datapoint represents high expression example from A. (C) Quantification showing the number of EB1 comets/µm² for cells with different StableMARK intensities (each dot represents a single cell, n = 115, N = 1; see Fig. 4 for replicate). Solid green line + dashed lines indicated mean ± SD of amount of EB1 comets/µm² for non-StableMARK-expressing cells (n = 99). (D) Graph depicting the intracellular concentration of StableMARK measured using FCS in cells classified by eye as low, medium, or high expressing. Median (line) and averages per independent experiment (colored dots) are shown, representing data from 16 to 20 cells per condition from four independent experiments. (E) Classification of mitochondria distribution at different levels of StableMARK expression (no expression, n = 124 cells; StableMARK expressing, n = 101 cells [low, n = 58 cells; medium, n = 32 cells; high, n = 11 cells]). Scale bar, 20 µm (A).