Figure S2.
Validation of the RPE1 NuMA KO line stably expressing NuMA-Bonsai-EGFP and estimate of NuMA-EGFP intensity in cells used in the FRAP analysis. (A) Normalized NuMA intensity in uninduced NuMA KO RPE1 cells (control), NuMA KO cells, and NuMA KO cells overexpressing the rescue constructs NuMA-FL-EGFP and NuMA-Bonsai-EGFP. An antibody against the N terminus was used in order to detect both endogenous and overexpressed protein. Plot shows mean ± SD n = 38 (control), 35 (NuMA KO), 21 (NuMA KO + NuMA-FL-EGFP), and 18 (NuMA KO + NuMA-Bonsai-EGFP) cells. Two-sided unpaired t test: *, P < 0.02. (B) Western blot of endogenous NuMA and NuMA-Bonsai-EGFP in uninduced NuMA KO RPE1 cells (control) and 96 h NuMA KO cells stably overexpressing NuMA-Bonsai-EGFP. (C) Percentage of cells with different nuclear phenotypes at 0 h, 24 h, 48 h, 72 h, and 96 h of endogenous NuMA KO in cells stably overexpressing NuMA-Bonsai-EGFP and fixed for immunofluorescence as in Fig. 1 B. n = 205 (0 h), 177 (24 h), 181 (48 h), 181 (72 h), and 177 (96 h) cells. (D) Chromosome segregation defects in live control and NuMA KO cells stably overexpressing Bonsai-NuMA-EGFP. Normal segregation indicates anaphase progression without lagging chromosomes or anaphase bridges. n = 8 (control) and 17 (NuMA KO) cells. 1 out of 17 NuMA KO cells exited mitosis with a defective nucleus, and without completing anaphase, and was scored as segregation defective. (E) NuMA-FL-EGFP, NuMA-Bonsai-EGFP, and NuMA-NC-EGFP fluorescence intensity in the cells analyzed in the FRAP experiment presented in Fig. 3, D and E. Plot shows mean ± SD n = 11 (NuMA-FL-EGFP), 23 (NuMA-Bonsai-EGFP), and 12 (NuMA-NC-EGFP). Two-sided Mann–Whitney test: ns, nonsignificant. (F) Fast recovery halftime as a function of NuMA-FL-EGFP, NuMA-Bonsai-EGFP, and NuMA-NC-EGFP intensity in the cells from Fig. 3, D and E. n = 11 (NuMA-FL-EGFP), 23 (NuMA-Bonsai-EGFP), and 12 (NuMA-NC-EGFP). MN, micronuclei; and WT, wild-type.

Validation of the RPE1 NuMA KO line stably expressing NuMA-Bonsai-EGFP and estimate of NuMA-EGFP intensity in cells used in the FRAP analysis. (A) Normalized NuMA intensity in uninduced NuMA KO RPE1 cells (control), NuMA KO cells, and NuMA KO cells overexpressing the rescue constructs NuMA-FL-EGFP and NuMA-Bonsai-EGFP. An antibody against the N terminus was used in order to detect both endogenous and overexpressed protein. Plot shows mean ± SD n = 38 (control), 35 (NuMA KO), 21 (NuMA KO + NuMA-FL-EGFP), and 18 (NuMA KO + NuMA-Bonsai-EGFP) cells. Two-sided unpaired t test: *, P < 0.02. (B) Western blot of endogenous NuMA and NuMA-Bonsai-EGFP in uninduced NuMA KO RPE1 cells (control) and 96 h NuMA KO cells stably overexpressing NuMA-Bonsai-EGFP. (C) Percentage of cells with different nuclear phenotypes at 0 h, 24 h, 48 h, 72 h, and 96 h of endogenous NuMA KO in cells stably overexpressing NuMA-Bonsai-EGFP and fixed for immunofluorescence as in Fig. 1 B. n = 205 (0 h), 177 (24 h), 181 (48 h), 181 (72 h), and 177 (96 h) cells. (D) Chromosome segregation defects in live control and NuMA KO cells stably overexpressing Bonsai-NuMA-EGFP. Normal segregation indicates anaphase progression without lagging chromosomes or anaphase bridges. n = 8 (control) and 17 (NuMA KO) cells. 1 out of 17 NuMA KO cells exited mitosis with a defective nucleus, and without completing anaphase, and was scored as segregation defective. (E) NuMA-FL-EGFP, NuMA-Bonsai-EGFP, and NuMA-NC-EGFP fluorescence intensity in the cells analyzed in the FRAP experiment presented in Fig. 3, D and E. Plot shows mean ± SD n = 11 (NuMA-FL-EGFP), 23 (NuMA-Bonsai-EGFP), and 12 (NuMA-NC-EGFP). Two-sided Mann–Whitney test: ns, nonsignificant. (F) Fast recovery halftime as a function of NuMA-FL-EGFP, NuMA-Bonsai-EGFP, and NuMA-NC-EGFP intensity in the cells from Fig. 3, D and E. n = 11 (NuMA-FL-EGFP), 23 (NuMA-Bonsai-EGFP), and 12 (NuMA-NC-EGFP). MN, micronuclei; and WT, wild-type.

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