Fig. 6: Mis12C-Ndc80C interaction is dispensable in human cells.

a Schematic representation of the KMN network on CENP-T in chicken and human cells. Chicken has KMN-N on CENP-T. While cKO-Dsn1/Dsn1^∆326-349 cells (KMN-N) died, expression of CENP-T^2X(1-90) in these cells (KMN-N-N) rescued growth deficiency. In human cells, KMN-N-N is on CENP-T. The phenotype of cKO-Dsn1 lacking the Mis12C-Ndc80C interaction (cKO-hDsn1/hDsn1^∆325-356 cell: KMN-N-N) will be tested. The KMN network on the CENP-C pathway in chicken and human cells is also presented. Both gDsn1^∆326-349 and hDsn1^∆325-356 do not bind to Ndc80C on the CENP-C pathway. b Immunoprecipitation with anti-RFP in AID-based cKO-hDsn1 cells expressing either mScarlet-hDsn1^WT or -hDsn1^∆325-356 in the presence of nocodazole for 24 h and IAA for last 2 h. Samples were analyzed by anti-Dsn1, and -Ndc80 antibodies. c Ndc80 levels at kinetochores in cKO-hDsn1 cells expressing mScarlet-hDsn1^WT or -hDsn1^∆325-356 in the presence of IAA for 2 h. DNA was stained with DAPI. Ndc80 was stained with an anti-Ndc80 antibody (red). CENP-T was stained with an anti-CENP-T antibody (green). None indicates parental cKO-hDsn1 (AID) cells. Error bars indicate the mean and standard deviation. p values were calculated by one-way ANOVA followed by Tukey’s test. d Tubulin staining in cKO-hDsn1 cells expressing hDsn1^WT or hDsn1^∆325-356 in the presence of IAA for 2 h. Kinetochores were stained with an anti-human CENP-A antibody (red). The insets present the kinetochore-microtubule bindings. DNA was stained with DAPI. e Images of mitotic chromosomes stained with anti-CENP-A in cKO-hDsn1 cells (None) expressing hDsn1^WT or hDsn1^∆325-356 in the presence of IAA for 2 h. DNA was stained with DAPI. The graph summarizes the percentage of cells with misaligned chromosomes in each line. Error bars indicate the mean and standard deviation. p values were calculated as in c. f Images of anaphase for cKO-hDsn1 cells (None) expressing hDsn1^WT or hDsn1^∆325-356 in the presence of IAA for 2 h. DNA was stained with DAPI. The graph summarizes the percentage of cells with abnormal anaphase in each line. Error bars indicate the mean and standard deviation. p values were calculated as in c. g The growth of cKO-hDsn1 cells expressing mScarlet-hDsn1^WT or -hDsn1^∆325-356 in the absence or presence of IAA.