Fig. 4: FOXJ1 depletion impairs MT dynamic.

A Immunoblot analysis of acetylated α-tubulin (K40) in DU145 cells expressing control shRNA (Neg) or independent FOXJ1 shRNAs (shRNA_01, shRNA_02, shRNA_03) treated with docetaxel (0, 50, or 100 nM) for 24 h. Band intensities were quantified by densitometry, normalized to GAPDH, and expressed as fold change relative to the corresponding negative control and indicated numerically below each band. The blot is representative of three independent experiments. B Grayscale images of EB1-EGFP–expressing DU145 shNeg or FOXJ1-knockdown (shRNA_01) cells, imaged live under basal conditions or following taxane treatment. EB1 comets marking growing MT plus ends are pseudo-colored to aid visualization and tracking; acquisition and analysis parameters (exposure, frame rate, denoising/background subtraction, and detection thresholds) were held constant within each experiment across conditions. The images are representative of three independent experiments. C EB1 comet number per cell in control versus treated cells was determined in an experiment that is representative of three biological repeats. Ten cells per condition were quantified; each dot (untreated) or triangle (taxane-treated) represents one cell. The center line denotes the mean and error bars indicate SD. D EB1 comet length in control versus treated cells was determined in an experiment that is representative of three biological repeats. Comet length was quantified from 10 cells per condition; each data point represents an individual comet (n ≈ 250), with dashed lines indicating mean ± SD. Mean comet length was 1.83 µm in shNeg cells and 1.59 µm in shRNA_01 cells. E EB1 comet growth speed (µm/sec) was quantified from time-lapse imaging of EB1 comet length in control versus treated cells in an experiment that is representative of three biological repeats. Each point represents an individual comet (n = 450–500 comets per condition), shown to visualize distribution breadth; horizontal bars denote mean ± SD. Mean speeds were 0.48 µm/s (untreated shNeg) vs 0.45 µm/s (untreated shRNA_01), decreasing to 0.23 µm/s (taxane-treated shNeg) and 0.20 µm/s (taxane-treated shRNA_01). F EB1 comet trajectory was mapped in control versus treated cells in an experiment that is representative of three biological repeats. Panels (left to right) show untreated shNeg, untreated shRNA_01, taxane-treated shNeg, and taxane-treated shRNA_01 cells. Top: maximum-intensity projections of EB1 trajectories generated from 10-s time-lapse sequences. Trajectories were generated from time-lapse imaging acquired over a 10-second interval and overlaid to visualize microtubule plus end growth dynamics. Tracks are color-coded using a continuous color spectrum to represent time progression, with blue indicating the initial position of each EB1 comet and red indicating the final position (see color timelapse bar for each panel). Bottom: representative frames with detected comets pseudo-colored to indicate individual tracks; corresponding trajectory traces or path lengths (µm) are shown below. The x-axis represents time (s) and the y-axis displays the path length (µm) for each EB1 trajectory. Maximum trajectory lengths reached 12 µm (untreated shNeg) and 9.5 µm (untreated shRNA_01), decreasing to 5 µm and 4.5 µm after taxane treatment, respectively. G Quantitative representation of the results shown in F. EB1 trajectory length (µm) was quantified from time-lapse imaging (450–500 comets per condition). Mean trajectory lengths were 13.30 µm (untreated shNeg) vs 11.96 µm (untreated shRNA_01), decreasing to 6.29 µm (taxane-treated shNeg) and 5.70 µm (taxane-treated shRNA_01). Source data are provided as a Source Data file.