MyTH4 domain expression increases junctional pCdk1 staining. (A) Uninjected control stage 10 embryos, or those expressing eGFP-MyTH4 or eGFP-MyTH4-DD or treated with 25 µM PD166285 for 30 min, were fixed and immunostained with antibodies against α-tubulin (MT, red) and pCdk1 (green). Bar, 20 µm. (B) Quantification of junctional pCdk1 signal in uninjected control embryos, or those expressing MyTH4 or MyTH4-DD or incubated for 30 min in 25 µM PD166285. y axis represents fold change in junctional pCdk1 relative to controls within the same experiment. The red line represents the mean control pCdk1 at junctions, defined as 1. A gray dot represents a single embryo, the blue dot the mean and the blue bars the 95% confidence interval. n = 10–15. All are significantly different from control (from left to right: P = 0.0004; P =0.0001; P =0.003). (C) Embryos were microinjected at the two-cell stage with mRNA encoding either GFP-MyTH4 (GFP-4) or GFP-MyTH4-DD (GFP-4-DD). At stage 10, whole-cell extracts were prepared and immunoblotted for α-tubulin (red) and pCdk1 (green). The numbers at the bottom of the blot indicate relative levels of pCdk1 in this image. Numbers on the left side indicate molecular weight in kilodaltons. (D) Stage 10 control or eGFP-MyTH4-expressing embryos were incubated for 0 or 30 min in 25 µM PD166285. The embryos were then fixed and stained for microtubules (anti–α-tubulin) and DNA (DAPI or TO-PRO3). The percentage of metaphase and anaphase cells were counted for each field of epithelium taken at 40×. In the plot, a gray dot represents a single embryo, the blue dot the mean, and the blue bars the 95% confidence interval. n = 10–20 embryos from four different experiments.