Figure 2.
Figure 2. Spindle checkpoint failure in CDKN3 siRNA-treated cells is not caused by off-target RNAi effects. (A) Overexpression of siRNA-resistant GFP-CDKN3 rescues the SAC failure phenotype in cells transfected with CDKN3 siRNA. Phenotype rescue in CDKN3 knockdown cells transfected with siRNA-resistant GFP-CDKN3 is indicated by mitotic arrest in taxol (red). No phenotype rescue and minimal GFP fluorescence were observed in CDKN3 siRNA cells transfected with siRNA-sensitive GFP-CDKN3 construct (top). Transfection with either GFP-CDKN3 construct resulted in a similar level of GFP-CDKN3 expression in cells pretransfected with control siRNA (bottom two rows). (B) Quantification of the rescue experiment. Error bars indicate SEM. n = 3 experiments; P < 0.0001 in t test. (C) Validated siRNAs against CDKN3 recapitulate the SAC failure phenotype. (D) Quantification of the SAC failure phenotype induced by three CDKN3 siRNAs shown in C. n = 9, P < 0.0001 in one-way ANOVA. (E) Validation of CDKN3 knockdown by four separate siRNAs in quantitative Western blots. n = 6, P < 0.0001 in one-way ANOVA. Note the normal CDKN3 signal in cells transfected with MAD2 siRNA. (F) Cells transfected with four separate CDKN3 siRNAs express normal MAD2 level. The MAD2/actin ratio was quantified by Western blotting. n = 6, (not significant) n.s. in one-way ANOVA for each CDKN3 siRNA. Note the MAD2 knockdown (P = 0.0025) in cells transfected with positive control MAD2 siRNA. (G) Normal localization of endogenous MAD2 on prometaphase kinetochores in cells transfected with CDKN3 siRNAs. Note the lack of MAD2 signal in cells transfected with validated MAD2 siRNA. (H) Quantification of MAD2-positive prometaphase kinetochores in cells transfected with CDKN3 siRNAs and MAD2 siRNAs. P < 0.0001 in ANOVA, n = 50 200-µm nuclear cross sections per siRNA. (I) MAD2 overexpression does not rescue the SAC failure phenotype induced by CDKN3 siRNA. Note the similar nuclear morphology of cells transfected with CDKN3 siRNA alone compared with CDKN3 siRNA followed by MAD2 overexpression. (J) The SAC failure in CDKN3 siRNA cells is not rescued by ectopic overexpression of MAD2. n = 10; n.s. in t test. (K) Expression of Flag-MAD2 in cells transfected with the construct used in rescue experiments (I and J).

Spindle checkpoint failure in CDKN3 siRNA-treated cells is not caused by off-target RNAi effects. (A) Overexpression of siRNA-resistant GFP-CDKN3 rescues the SAC failure phenotype in cells transfected with CDKN3 siRNA. Phenotype rescue in CDKN3 knockdown cells transfected with siRNA-resistant GFP-CDKN3 is indicated by mitotic arrest in taxol (red). No phenotype rescue and minimal GFP fluorescence were observed in CDKN3 siRNA cells transfected with siRNA-sensitive GFP-CDKN3 construct (top). Transfection with either GFP-CDKN3 construct resulted in a similar level of GFP-CDKN3 expression in cells pretransfected with control siRNA (bottom two rows). (B) Quantification of the rescue experiment. Error bars indicate SEM. n = 3 experiments; P < 0.0001 in t test. (C) Validated siRNAs against CDKN3 recapitulate the SAC failure phenotype. (D) Quantification of the SAC failure phenotype induced by three CDKN3 siRNAs shown in C. n = 9, P < 0.0001 in one-way ANOVA. (E) Validation of CDKN3 knockdown by four separate siRNAs in quantitative Western blots. n = 6, P < 0.0001 in one-way ANOVA. Note the normal CDKN3 signal in cells transfected with MAD2 siRNA. (F) Cells transfected with four separate CDKN3 siRNAs express normal MAD2 level. The MAD2/actin ratio was quantified by Western blotting. n = 6, (not significant) n.s. in one-way ANOVA for each CDKN3 siRNA. Note the MAD2 knockdown (P = 0.0025) in cells transfected with positive control MAD2 siRNA. (G) Normal localization of endogenous MAD2 on prometaphase kinetochores in cells transfected with CDKN3 siRNAs. Note the lack of MAD2 signal in cells transfected with validated MAD2 siRNA. (H) Quantification of MAD2-positive prometaphase kinetochores in cells transfected with CDKN3 siRNAs and MAD2 siRNAs. P < 0.0001 in ANOVA, n = 50 200-µm nuclear cross sections per siRNA. (I) MAD2 overexpression does not rescue the SAC failure phenotype induced by CDKN3 siRNA. Note the similar nuclear morphology of cells transfected with CDKN3 siRNA alone compared with CDKN3 siRNA followed by MAD2 overexpression. (J) The SAC failure in CDKN3 siRNA cells is not rescued by ectopic overexpression of MAD2. n = 10; n.s. in t test. (K) Expression of Flag-MAD2 in cells transfected with the construct used in rescue experiments (I and J).

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