Fig 2: Analysis of NSMCE2 null cell growth characteristics: (A) Phase contrast images of WT and NSMCE2 null cells. The latter shows a large number of vacuoles in the cytoplasm (arrow). Bar = 20 µm; (B) Average cell cycle duration of WT and NSMCE2 null cells over multiple generations observed by live cell imaging. Data are presented as mean ± standard error of mean (SEM), n = 3; (C) Cell cycle phase distribution analysis of WT and NSMCE2 null cells by DNA histograms shows a 10% increase NSMCE2 null cells in G0-1; (D) G0-1 phase depletion by colcemid treatment. WT and NSMCE2 null cells were treated with colcemid for 0–96 h. In NSMCE2 null cells, a fraction of cells remained in G0-1 even after 96 h; (E) Quantification for depletion of G0-1 cells with the time of colcemid treatment. Data are presented as mean ± SEM, n = 3; (F) Western blot analysis of SMC5 and SMC6 proteins in WT and NSMCE2 null cells. ß-Actin was used as a loading control.

Fig 3: KDM2A facilitates SENP6-mediated ALT telomere de-SUMOylation.a Representative immuno-FISH images of multitelomere clusters in control or SMC5 siRNA transfected mitotic ALT#1 cells expressing sgCtrl or sgK#1. The mitotic (M) cells were prepared from G2-synchronized cells after 1 h release from the CDK1 inhibitor. The arrows denote abnormal mitotic telomere cluster foci. b Percentages of mitotic cells containing =3 telomere cluster foci are expressed as mean ± s.e.m. of three independent experiments; two-tailed unpaired t-test. c Western blot analysis of SMC5 expression in whole-cell lysates prepared from the siCtrl or siSMC5-transfected ALT#1 cells. d Representative immuno-FISH images of telomere SUMOylation in sgCtrl or sgK#1-transduced ALT#1 cells. The mitotic (M) cells were from G2-synchronized cells upon 1 h release from the CDK1 inhibitor. The arrows denote SUMO2/3-associated telomere foci. e Percentages of cells containing =2 SUMO-associated telomere foci. Data were expressed as mean ± s.e.m. of three independent experiments; two-tailed unpaired t-test. f Competition-based proliferation assay of the indicated SENP6-targeted sgRNAs (sgSEN#1 and sgSEN#2) on the fitness of IMR90-T, ALT#1 and Saos2 cells. The data are expressed as mean ± s.e.m. of three independent experiments; two-tailed paired t-test. g Western blot analysis of SENP6 in whole-cell lysates prepared from sgCtrl, sgSEN#1 or sgSEN#2-transduced ALT#1 cells. h Immuno-FISH analysis of telomere SUMOylation in mitotic ALT#1 cells transduced with sgCtrl, sgSEN#1, or sgSEN#2. The mitotic cells were from G2-synchronized cells upon 1 h release from the CDK1 inhibitor. The arrows denote SUMO2/3-associated telomere foci. i Percentages of mitotic cells containing =2 SUMO-associated telomere foci are expressed as mean ± s.e.m. of three independent experiments; two-tailed unpaired t-test. j Western blot analysis of Flag and ACTB (loading control) in whole-cell lysates prepared from the indicated ALT#1 cells transduced with Flag-tagged SENP6C1030A mutant. k Immuno-FISH analysis of telomere and SENP6C1030A association in Flag-SENP6C1030A -expressing ALT#1 cells transduced with sgCtrl or sgK#1. The arrows denote SENP6C1030A-associated telomere foci. l Percentages of cells containing =3 Flag-SENP6C1030A-associated telomere foci are expressed as mean ± s.e.m. of three independent experiments; two-tailed unpaired t-test. m A model illustrating the key events in the ALT pathway. Source data are provided as a Source Data file.