Fig 2: Relationship of cell volume to GAPDH and MDH1 cell v 0 for oocytes at three stages of development. The data for GAPDH (red) and MDH1 (blue) are shown in the left and right panels, respectively. When considered as a temporal series, the data are consistent with developmental regulation of the strength of the relationship between cell volume and cell v 0. The pattern is similar for GAPDH and MDH1: a robust increase of correlation strength from stage II to stage IV, followed by a stronger decline from stage IV to VI (compare top, middle, and bottom plots for each enzyme). Equations for the lines of best fit are presented in Table S2D. Note that the R 2 values shown here for stage VI oocytes of animal 1 approach those determined for stage VI oocytes of animal 3 (Figure 3D).

Fig 3: Cleavage of caspase-6 substrate candidates was confirmed by in vivo cleavage assay. HeLa cells were treated with 1 µM STS for 24 hr in the presence or absence of the 100 µM caspase inhibitors (Z-VEID-FMK; caspase-6 inhibitor, Z-DEVD-FMK; caspase-3/-7 inhibitor, Z-VAD-FMK; pan-caspase inhibitor) pretreated to cells for 3 hr. After 24 hr, cells were harvested and lysed. Cell lysates were analyzed by immunoblotting with the specific antibodies against (A) HAUSP, Kinesin5B, GEP100, SDCCAG3, PARD3, Lamin A/C (Cl, cleaved Lamin A/C), and PARP (Cl, cleaved PARP); and (B) CLK3, ADH4, MDH1, Lamin A/C, and PARP. A coomassie blue stain of PVDF membrane was the loading control. The representative blot of at least two independent experiments is shown.

Fig 4: Cleavage of caspase-6 substrate candidates was confirmed by in vitro cleavage assay. (A) HepG2 extract was incubated with active caspase-6 (10 U) or inactive capsase-6 (10 U) in the presence or absence of 30 µM Z-VEID-FMK. The reaction mixture was analyzed by immunoblotting with the specific antibodies against (A) HAUSP, Kinesin5B, GEP100, SDCCAG3, PARD3 and Lamin A/C. (B) HepG2 extract was incubated with increasing amount of active caspase-6 (5, 10, 15, 20 U) or inactive capsase-6 (20 U). The reaction mixture was analyzed by immunoblotting with the specific antibodies against CLK3, ADH4, MDH1, and Lamin A/C (Cl, cleaved Lamin A/C). Lamin A/C cleavage was used as a positive control. The asterisk indicates a nonspecific band. The representative blot of at least two independent experiments is shown. 1 U is the amount of enzyme required to cleave 1nmole of caspase substrate completely in 1 hr.

Fig 5: Variability of GAPDH and MDH1 cell v 0 in individual oocytes of three types. (A) cell v 0 was determined for GAPDH (red) and MDH1 (blue) in individual stage II, IV, and VI oocytes from two animals (top two panels; each box and whisker plot shows the data for 18 cells). v 0 for GAPDH and MDH1 was also determined for control ensemble homogenates of the same oocyte types (technical replicates in bottom panel). Variability as relative standard deviation (RSD shaded in gray) is higher for each set of 18 individual oocytes (top panels) than for variability of activity associated with repeat sampling of the corresponding (stage-matched) technical replicate homogenates. For each type of oocyte, the variability of enzyme activity between individual cells was higher than the variability between replicate samples from the corresponding control homogenate. For example, the RSD for GAPDH in the 18 stage II cells of animal 1 was 27.7% (top panel, left-most plot); the RSD for the corresponding technical replicate control was 1.57% (bottom panel, left-most plot). (B) Plots of the relative standard deviation (RSD) associated with assay of GAPDH and MDH1 activity in individual cells (cellRSD, left two panels) and in repeat samples of technical replicate homogenates (trRSD, right panel). This is a representation of the RSD data in A.