Interplay between BB alignment and orientation. (A) Schematic drawing (top) and dual-color immunofluorescence for Odf2 and centriolin (middle and bottom), which marked the BB as a ring (green) and BF as a dot (magenta), respectively. Original image is shown in Fig. S2 A. (B) Representative immunofluorescence micrographs of each pattern, with an illustration showing a line from the center of the green ring to the center of the magenta dot. (C) Angle histograms of BB directions from representative cells in each pattern. (D) Boxplot of the dependency of the index of orientation (Io) on the ALI day (n = 10 cells/day). On each box, the red segment indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the lowest and highest data points, respectively. (E) Correlation between the linearity of the alignments and the uniformity of the directions during MCC differentiation. Each plotted point (black circle) represents a single cell in terms of its degree of BB alignment (horizontal axis, Ia) and orientation (vertical axis, Io). Histograms of Ia and Io are shown by gray bars. Immunofluorescence images of 100 cells with a total of 28,501 BB–BF pairs were analyzed. Bars, 700 nm.