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Supplement Figure 1.
GV GVBD MI MII
CapZ β2
Merge
DNA CapZ β2
A
DIC GFP-CapZ β2 Merge
250ng
500ng
B CapZ β2-GFP
Journal of Cell Science | Supplementary Material
Supplement Figure 2.
A B Control
early MI
late MI
RNAiCapZαβ
Control RNAiCapZαβ
n=10
n=20
N.S.
Pha
lloid
in In
tens
ity(A
.U.)
DNA Actin
Tubulin
Journal of Cell Science | Supplementary Material
Journal of Cell Science | Supplementary Material
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
Journal of Cell Science | Supplementary Material
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:
Journal of Cell Science | Supplementary Material
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-Tubulin-
eGFP-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.
!
Journal of Cell Science | Supplementary Material
Movie 1.
Movie 2.
Movie 3.
Journal of Cell Science | Supplementary Material
Movie 4.
Movie 5.
Movie 6.
Journal of Cell Science | Supplementary Material
Table S1. Primers used in this study
Gene Accession no. Primer sequence Use of the primer
CapZ�1 NM_009797.2
5’-ATGGCCGACTTTGAGGATCG-3’ qPCR (Forward)
5’-CAGAGCACTGTCACACGAC-3’ qPCR (Reverse)
5’-TAATACGACTCACTATAGGGAGACCAC TGGTGACTTGGTAACAGCA-3’
dsRNA (Forward)
5’-TAATACGACTCACTATAGGGAGACCAC GATTTTGGTGCGGGTAACTG-3’
dsRNA (Reverse)
CapZ�2 NM_001271405.1
5’-AGGCAGCCTAACCAGACAGA-3’ qPCR (Forward)
5’-CCTCCACCAGGTCGTTCTTA-3’ qPCR (Reverse)
5’-TAATACGACTCACTATAGGGAGACCAC GGTGGGCAAGGATTACCTTT-3’
dsRNA (Forward)
5’-TAATACGACTCACTATAGGGAGACCAC CCTCCACCAGGTCGTTCTTA-3’
dsRNA (Reverse)
dsRNA; double-stranded RNA; qPCR, quantitative PCR.
Journal of Cell Science | Supplementary Material
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