Image analysis pipeline for the determination of subcellular distribution in polarized cells. (A) Representative image of a hemocyte expressing LifeAct-GFP stained with LysoTracker probe and treated with 15 µg/ml of 20-hydroxyecdysone to promote polarization. (B) The channel corresponding to actin cytoskeleton was used to obtain a mask for signal intensity measurement. (C) A three-channel image (two original channels + mask) was used to run the Radial Reslice plugin. The white dashed line represents the adjusted circumference around the center of the cell. The first radial signal to be obtained is at the center of the extended lamella (0°) as the program runs, it completes the 360° of the cell clockwise (white round arrow). (D) After the plugin was run, the radial signals of all channels were obtained (360° each). The radial signals from the masks were used to define regions of interest (ROIs) to measure signal intensity of the two original channels. The ROIs were converted to metric units, corresponding to the local length of the cell radius (r). (E) Obtained values were normalized so that in all cases, average signal intensity = 1. Normalized values were plotted to demonstrate graphical distribution of signal in all 360° of the cells. (F) All intensity values from E corresponding to the rear 90° (135°–225°) were compared with the front 90° (315°–360°, 0°–45°). A.U., arbitrary units; Ecd, ecdysone. In this type of analysis, two-way ANOVA with Sidak’s multiple comparisons test were performed (*, P < 0.05). n.s., not significant. (F’) Polarized cell in which the front (F) and rear (R) signals plotted in F are highlighted in blue and red, respectively.  The white dashed line represents the adjusted circumference around the center of the cell.