Disruption of Sbf and Rab35 function leads to unbalanced forces phenotype. (A) Definition of cell categories (1–5, colored rows) based on distance from ventral midline. (B) Quantification of absolute rate of cell area change of different cell categories defined in A in control, Sbf shRNA, and Rab35 shRNA background. Control cell numbers quantified from category 1 to 5 are n = 64, 65, 63, 31, and 23, respectively; Sbf shRNA cell numbers are n = 93, 92, 76, 41, and 36, respectively; and Rab35 shRNA cell numbers are n = 87, 92, 88, 91, and 65, respectively. (C) Cell outlines (Spider:GFP) in control, Sbf shRNA, and Rab35 shRNA backgrounds by live imaging. Red dots and black dots mark large cells and small cells, respectively. Spider:GFP is heterozygous in Sbf and Rab35 images. (D) Aspect ratio of cell shapes of control, Sbf shRNA, and Rab35 shRNA embryos. n = 152 (control), 196 (Sbf shRNA) and 216 (Rab35 shRNA) cells. (E) Standard deviation of cell areas in control, Sbf shRNA and Rab35 shRNA embryos. n = 379 (t = 0, control), 359 (t = terminal, control), 404 (t = 0, Sbf shRNA), 374 (t = terminal, Sbf shRNA), 419 (t = 0, Rab35 shRNA), and 398 (t = terminal, Rab35 shRNA) cells. (F) The number of cells >40 µm². n = 324 (control), 79 (Rab35 shRNA), and 89 (Sbf shRNA) cells. (G) Quantification of contractile steps followed by expansion steps in big cells. (H) The number of steps per minute in big cells in control, Sbf shRNA, and Rab35 shRNA embryos. (G and H) n = 33 (control), 23 (Sbf shRNA), and 29 (Rab35 shRNA) cells. (I) The quantification of change of average steps in big cells. n = 174 (control), 182 (Sbf shRNA), and 347 (Rab35 shRNA) steps. Scale bar represents 5 μm. Error bars indicate standard errors. Statistical significance was calculated using a Mann–Whitney U test (B) and Student’s t test (E–I). *, P < 0.05; **, P < 0.005; ***, P < 0.0005. In A and C, embryos are oriented with anterior up and posterior down.