Figure 6.
Figure 6. Depletion of MAD2L2 leads to premature binding of CDH1 to the APC/C in prometaphase. (A) CDH1 interacts with the APC/C within 60 min after nocodazole release. Immunoprecipitation with anti-CDC27 from U2OS cells arrested in nocodazole and at 30, 60, and 90 min after release blotted for CDH1 to monitor the association of CDH1 with the APC/C. (B) Silencing of MAD2L2 leads to premature association of CDH1 with CDC27. (C) Complementation of the premature association of CDH1 with CDC27. The cells in this experiment all stably express hMAD2L2-myc. siMAD2L2 silences both the endogenous MAD2L2 and the transgenic MAD2L2-myc. The siMAD2L2 3′UTR only silences the endogenous MAD2L2, leaving the MAD2L2-myc expressed. See also Fig. 1 C. (D) Quantification of the premature association of CDH1 with CDC27 in MAD2L2-depleted cells. The amount of CDH1 immunoprecipitated with CDC27 was normalized to the IgG signal for each immunoprecipitation and the ratio of the amount of CDH1 pulled down in nocodazole (time = 0) and at 60 min after release was calculated. The constitutive association of CDH1 with CDC27 in prometaphase in MAD2L2-silenced cells results in an increase in this ratio, which approaches 1. “n” represents the number of independent experiments. Error bars = SEM; P-value calculated with unpaired, two-tailed t test assuming equal variance. The additional blots contributing to this analysis are shown in Fig. S1 D. (E) Depletion of CDH1 prevents the rapid mitotic exit seen in cells lacking MAD2L2. The percentage of cells in G1 was assessed 60 min after nocodazole release. “n” represents the number of independent experiments. Error bars = SEM; p, unpaired t test. The control siRNA and siMAD2L2 data are reproduced from Fig. 1 D for comparison. The effectiveness of the siRNA protocol is illustrated in the panel on the right. (F) Silencing of MAD2L2, but not REV3, can rescue the mitotic delay in CDC20-depleted cells. Quantification of the time taken for control (Control siRNA; solid black line), MAD2L2-depleted (siMAD2L2; solid red line), REV3-depleted (siREV3; solid blue line), CDC20-depleted (siCDC20, dashed dark gray line), CDC20- and MAD2L2-depleted (siCDC20 + siMAD2L2, dashed red line), and CDC20- and REV3-depleted (siCDC20 + siREV3, dashed blue line) U2OS cells expressing mCherry-H2B to complete mitosis, assessed by time-lapse video microscopy (1 frame every 5 min). The plot shows the cumulative percentage of cells that completed mitosis, measured from NEBD to cytokinesis. “n” represents the number of cells examined, collected from at least three independent experiments. P-value for siCDC20 and siCDC20 + siREV3 vs. Control siRNA, siMAD2L2, and siMAD2L2 + siCDC20 < 1 × 10−4.

Depletion of MAD2L2 leads to premature binding of CDH1 to the APC/C in prometaphase. (A) CDH1 interacts with the APC/C within 60 min after nocodazole release. Immunoprecipitation with anti-CDC27 from U2OS cells arrested in nocodazole and at 30, 60, and 90 min after release blotted for CDH1 to monitor the association of CDH1 with the APC/C. (B) Silencing of MAD2L2 leads to premature association of CDH1 with CDC27. (C) Complementation of the premature association of CDH1 with CDC27. The cells in this experiment all stably express hMAD2L2-myc. siMAD2L2 silences both the endogenous MAD2L2 and the transgenic MAD2L2-myc. The siMAD2L2 3′UTR only silences the endogenous MAD2L2, leaving the MAD2L2-myc expressed. See also Fig. 1 C. (D) Quantification of the premature association of CDH1 with CDC27 in MAD2L2-depleted cells. The amount of CDH1 immunoprecipitated with CDC27 was normalized to the IgG signal for each immunoprecipitation and the ratio of the amount of CDH1 pulled down in nocodazole (time = 0) and at 60 min after release was calculated. The constitutive association of CDH1 with CDC27 in prometaphase in MAD2L2-silenced cells results in an increase in this ratio, which approaches 1. “n” represents the number of independent experiments. Error bars = SEM; P-value calculated with unpaired, two-tailed t test assuming equal variance. The additional blots contributing to this analysis are shown in Fig. S1 D. (E) Depletion of CDH1 prevents the rapid mitotic exit seen in cells lacking MAD2L2. The percentage of cells in G1 was assessed 60 min after nocodazole release. “n” represents the number of independent experiments. Error bars = SEM; p, unpaired t test. The control siRNA and siMAD2L2 data are reproduced from Fig. 1 D for comparison. The effectiveness of the siRNA protocol is illustrated in the panel on the right. (F) Silencing of MAD2L2, but not REV3, can rescue the mitotic delay in CDC20-depleted cells. Quantification of the time taken for control (Control siRNA; solid black line), MAD2L2-depleted (siMAD2L2; solid red line), REV3-depleted (siREV3; solid blue line), CDC20-depleted (siCDC20, dashed dark gray line), CDC20- and MAD2L2-depleted (siCDC20 + siMAD2L2, dashed red line), and CDC20- and REV3-depleted (siCDC20 + siREV3, dashed blue line) U2OS cells expressing mCherry-H2B to complete mitosis, assessed by time-lapse video microscopy (1 frame every 5 min). The plot shows the cumulative percentage of cells that completed mitosis, measured from NEBD to cytokinesis. “n” represents the number of cells examined, collected from at least three independent experiments. P-value for siCDC20 and siCDC20 + siREV3 vs. Control siRNA, siMAD2L2, and siMAD2L2 + siCDC20 < 1 × 10−4.

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