Figure 7: The SCMC regulates F-actin through Cofilin.

(a) Immunoblots of egg and zygote lysates at similar stages from control and Tle6^Null female with Cofilin and pCofilin antibody. (b) Relative abundance (mean±s.e.m.) of pCofilin in control and SCMC^Null eggs and zygotes was determined by intensity of immunoblot from four independent experiments. Controls were set as 100%. (c) Western blot of pCofilin during mitotic zygotes (synchronous) from control and Tle6^Null females. (d) Co-IP of Normal (Norm) and Mater^Null egg lysates with TLE6 (α-TLE) or MATER (α-MAT) antibodies. Input, egg lysates from normal and Mater^Null females; IgG, normal rabbit IgG; Norm, normal egg lysates. (e) In situ Cofilin–FLOPED interactions were detected by PLA using their antibodies. Floped^Null Zygote (negative control); Scale bar, 20 μm. (f) Normal interphase zygotes were injected with buffer or mRNA of Myc-confilin-S3A (S3A), and cultured for 4–7 h. After fixation, embryos were mixed and stained with Myc antibody and phalloidin. Myc staining was used to distinguish zygotes from control (buffer) or embryos injected with S3A. Scale bar, 20 μm. (g) Relative immunofluorescent intensity (mean±s.e.m.) of subcortical F-actin in paired zygotes from control and zygotes expressing S3A, Myc-Cofilin-S3E (S3E) or treated with S3 peptides (S3). S3-treated embryo was distinguished from control by pre-staining with TLE6 antibody. Number of paired embryos for S3A, S3E and S3 was 17 (two experiments), 27 (two experiments) and 31 (six experiments), respectively. *P<0.05 indicates statistically significant differences using Student’s t-test; NS, no statistically significant differences. (h) Zygotes were treated as in f, but cultured to metaphase and stained. Scale bar, 20 μm. (i) Zygotes were treated as in g and cultured to metaphase. Spindle position (mean±s.e.m.) was calculated as in Fig. 4c. Embryo numbers were 33 (S3A, six experiments), 10 (S3E, two experiments) and 61 (S3, nine experiments). *P<0.05; ***P<0.001 indicates statistically significant differences using Student’s t-test. (j) Zygotes were treated as in g, and cultured to develop into two-cell embryos and stained. Scale bar, 20 μm. (k) Percent (mean±s.e.m.) of two-cell embryos with asymmetric division was calculated as in Fig. 4a after the treatment as in g (three independent samples).