The linker region and the C-terminus of ANI-1 are both required to restore NMY-2 asymmetry in the ring. (A) Total ring intensity of NMY-2::mKate in control and the indicated FLAG::ANI-1 expressing embryos at 50% constriction for indicated RNAi conditions. (B) End-on reconstruction of NMY-2::mKate at the cleavage plane for FLAG::ANI-1 expressing embryos at indicated % of ring constriction treated with ani-1(RNAi). White stars indicate the position of the leading edge. Scale bars is 5 µm. (C) Since fluorescence intensity is attenuated with increasing distance from the objective (Fig. S5 A), we quantified the leading and lagging edge fluorescence intensities on the left and right sides of the ring. To define the leading and the lagging edge the position of cytokinetic center (C_t) was assigned to the left or right half of the cleavage plane. The ring edge that was in the same half as the cytokinetic center was defined as the lagging edge. The fluorescence intensity was summed up at the leading or lagging edge (yellow) excluding the top and bottom regions (grey) of the ring. (D and E) Fluorescence intensity of NMY-2::mKate at the leading and the lagging edge (D) and the ratio of NMY-2::mKate intensities of the leading and lagging edge (E) for the indicated FLAG::ANI-1 expressing embryos at 50% ring constriction for indicated RNAi conditions. The fold increase of NMY-2::mKate at the leading and lagging edge after ani-1(RNAi) is highlighted with pink arrows. For all: P values were calculated using two-tailed Student’s t test or Mann–Whitney U test and are n.s. P > 0.05, *P < 0.05, **P < 0.01 and ***P < 0.001, n = number of embryos, and error bars are SEM.