A GV GVBD MI DNA CapZ β2 CapZ β2 Merge B DIC GFP-CapZ β2 Merge CapZ β2-gfp 250ng 500ng Supplement Figure 1. MII
A early MI late MI Control RNAi CapZαβ DNA Actin Tubulin B Phalloidin Intensity(A.U.) n=10 Control N.S. n=20 RNAi CapZαβ Supplement Figure 2.
Supplementary Data Supplement Figure 1. Localization of actin-capping protein (CP) during mouse oocyte maturation. (A) Subcellular localization of CapZβ2 during mouse oocyte meiotic maturation. Immunofluorescence staining was performed using an anti-capzβ2 antibody after methanol fixation. Blue: DNA; green: CapZβ2. Bar = 20 µm. (B) Localization of CapZβ2 in germinal vesicle (GV)-stage oocytes. GFP-CapZβ2 mrna was injected into the oocyte cytoplasm. Two different concentrations (250 and 500 ng/µl) of. GFP-CapZβ2 mrna were injected. Bar = 20 µm. Supplement Figure 2. Knockdown of actin-capping protein (CP) have no effect formation of the cortical actin and actin cap in maturing oocytes. (A) Cortical actin and actin cap formation by CP knockdown. After dsrna injection and maturation arrest to ensure CP was knocked down, maturation was resumed and samples were collected 7 h and 9 h later. Representative oocytes were immunostained for actin (red), β-tubulin (green), and DNA (blue). Bar = 20 μm. (B) Fluorescence intensity of Cortical actin labeling after injection of CapZα1 and CapZβ2 dsrna (CPKD) (Control: n = 10; RNAi: n = 20). N.S.: not statistically significant (p > 0.05).! Supplement Figure 3. Relationship between GFP fluorescence intensity and the polar body/oocyte diameter ratio in GFP-CapZβ2-overexpressing oocytes. GFP fluorescence intensity was measured in single oocytes (Y axis) and plotted against the ratio between the diameter of the polar body and that of the oocyte (X axis). Spindle migration speed in control GFP-expressing oocytes (A) and GFP-CapZβ2-overexpressing
oocytes (B).Maturing oocytes injected with the actin probe mcherry-utrch (red) and GFP-CapZβ2 (green) were imaged. DNA (blue) was stained with Hoechst 33342. The number of hours after the resumption of maturation is indicated in each frame. Bar = 20 μm. Supplementary Movies Movies 1 and 2. Time-lapse movie of an oocyte injected with α-tubulin-gfp (control). Images were taken 5 13 h after the resumption of maturation. The frame interval is 300 s and the total length of the movie is 28,800 s. Left: differential interference contrast (DIC), middle: spindle labeled with α-tubulin-gfp (green), and right: merged. Movies 3 and 4. Impaired spindle migration in an oocyte injected with CapZα1 and CapZβ2 double-stranded RNA (dsrna) and α-tubulin-egfp complementary RNA (crna). Images were taken 5.5 13.5 h after the resumption of maturation. The frame interval is 300 s and the total length of the movie is 28,800 s. Left: differential interference contrast (DIC), middle: CapZα1 and CapZβ2 dsrna and spindle labeled with α-tubulin-egfp (green), and right: merged. Movie 5. Symmetric division of an oocyte overexpressing GFP-CapZβ2. Images were taken 6 11 h after the resumption of maturation. Cytokinesis starts at 9 h and is completed at 11 h, which is earlier than in double-stranded RNA (dsrna)-injected oocytes. The frame interval is 300 s and the total length of the movie is 18,000 s. Left:
differential interference contrast (DIC), middle: GFP-CapZβ2 and H2B-mCherry, which labels chromatin, and right: merged. Movie 6. Early spindle migration and abnormal polar body protrusion in an oocyte overexpressing GFP-CapZβ2. Images were taken 6 11 h after the resumption of maturation, which was initiated by the removal of milrinone from the medium. Polar body extrusion is completed at 10 h, which is earlier than in control oocytes (α-tubulinegfp-injected) and double-stranded RNA (dsrna)-injected oocytes. Segregation of the polar body is observed at 10.5 h. The frame interval is 300 s and the total length of the movie is 18,000 s. Left: differential interference contrast (DIC), middle: GFP-CapZβ2 and H2B-mCherry, which labels chromatin, and right: merged.!
Movie 1. Movie 2. Movie 3.
Movie 4. Movie 5. Movie 6.
Table S1. Primers used in this study Gene Accession no. Primer sequence Use of the primer 5 -ATGGCCGACTTTGAGGATCG-3 qpcr (Forward) CapZ 1 NM_009797.2 5 -CAGAGCACTGTCACACGAC-3 5 -TAATACGACTCACTATAGGGAGACCAC TGGTGACTTGGTAACAGCA-3 qpcr (Reverse) dsrna (Forward) 5 -TAATACGACTCACTATAGGGAGACCAC GATTTTGGTGCGGGTAACTG-3 5 -AGGCAGCCTAACCAGACAGA-3 dsrna (Reverse) qpcr (Forward) CapZ 2 NM_001271405.1 5 -CCTCCACCAGGTCGTTCTTA-3 5 -TAATACGACTCACTATAGGGAGACCAC GGTGGGCAAGGATTACCTTT-3 5 -TAATACGACTCACTATAGGGAGACCAC CCTCCACCAGGTCGTTCTTA-3 dsrna; double-stranded RNA; qpcr, quantitative PCR. qpcr (Reverse) dsrna (Forward) dsrna (Reverse)