MARK2-YFP localizes as punctuate-striation pattern upon MARK2 inhibition. (a) Experimental regime and procedures. HeLa cells were exposed to Doxycycline 16 h before imaging. SiR-Actin (100 nM) and MARK2i (10 µM) or DMSO (solvent control) were added 30 min prior to imaging. (b) Deconvolved z-slice of 3D image stacks show MARK2-YFP (green) at the cell-substrate interface in WT treated with DMSO, MARK2i or in KD stained with SiR-Actin dye (red). White boxes show punctate foci pattern in WT (DMSO), punctate-striation pattern in WT (MARK2i) and long striation in KD. (c) Segmentation results of MARK2-YFP signal for quantification after manually outlining interphase cell boundaries. (d) The boxplots show eccentricity measurements for DMSO, MARK2i, MARK2i prolonged treatment (16 h) and KD. 0 represents a perfect circle and 1 a perfect line. For global group comparison, a generalized linear model (GLM) was fitted (non-normality was determined with Shapiro-Wilk test) followed by a post-hoc analysis for pair-wise comparison (Dunn’s post-hoc test) after Multiple-Comparison Kruskal–Wallis (MCKW) at a significant level of P < 0.01. n refers to the number of cells obtained from three experiments. Scale bars: 15 µm; 1 µm for inset.