Fig 1: Validate the binding interface between CENP-H and CENP-I, and the assembly mode of CENP-H/I/K ternary complex. ( A) Zoomed-in view of the binding interface between thCENP-HCT and ctCENP-INT. The interacting residues were drawn in stick and marked in number. (B) The relative binding activity of thCENP-H (WT and mutants) bound to GST-ctCENP-INT was assessed using GST pull-down assays. Error bars represent standard deviations, which were obtained from three independent experiments. The representative results of the pull-down assays were also available in Supplementary Figure S8A. (C) In vitro binding assays were performed to examine the binding activity among the ternary complex. Proteins with distinct combinations as indicated were mixed and GST pull-down assays were performed. The bead-bound proteins were resolved with SDS-PAGE and stained with Coomassie Blue. (D) In vitro binding assays were performed to assessing the effects of thCENP-H mutants on the formation of the fungal CENP-H/I/K ternary complex. Proteins with distinct combinations as indicated were mixed and GST pull-down assays were performed. The bead-bound proteins were resolved with SDS-PAGE and stained with Coomassie Blue. (E) Quantification of the relative binding activity between ctCENP-INT and thCENP-H (WT and mutants) in the presence of thCENP-K based on the results from lanes 5, 6 and 7 in (D). (F) Zoomed-in view of the binding interfaces of thCENP-HCT in the complex structure. The interacting resides in thCENP-H were drawn in stick and marked with numbers in red. And the counterpart residues in hsCENP-H were shown with numbers in black. ( G) HeLa Tet-On cells transfected with GFP-hsCENP-H (WT or mutants), non-tagged hsCENP-K and MYC-hsCENP-I. Cell lysates were treated with anti-GFP antibody. Immunoprecipitated samples were resolved with SDS-PAGE and blotted with the indicated antibodies. ( H) Quantification of the relative binding activity between hsCENP-I and hsCENP-H WT or mutants (upper panel), and between hsCENP-K and hsCENP-H WT or mutants (lower panel) based on the results from Figure 4G.
Fig 2: Mutating the interface residues of CENP-H abolished its centromeric localization and induced massive chromosome mis-alignment. (A and B) Representative images of interphase HeLa Tet-On cells treated with Luciferase and hsCENP-H siRNA. The centromeric localization of endogenous hsCENP-H (A) or hsCENP-I (B) was detected by indicated antibody. (C) Quantification of the centromeric intensities of hsCENP-H in (A) and hsCENP-I in (B) normalized to the ones of ACA (CREST). At least 10 cells (20 kinetochores per cell) were quantified for each condition. Mean ± SD (standard deviation) was shown here. (D) HeLa Tet-On cells transfected with RNAi-resistant GFP-hsCENP-I WT, L219AL233A and K234E/L238A were treated with hsCENP-H siRNA. Cells were briefly treated with MG132 before subjected to staining. Representative images of mitotic cells were shown here. (E and F) Quantification of the centromeric intensities of GFP-hsCENP-H (E) and hsCENP-I (F) normalized to the ones of ACA (CREST) kinetochore signal. At least 10 cells (20 kinetochores per cell) were quantified for each condition. Mean ± SD was shown here. (G) Quantification of mitotic cells with chromosome alignment defects for the experiments in (A, B and D). At least 50 cells were counted for each condition. Mean ± SD was shown here. (H) The assembly mode of CENP-H/I/K ternary complex among the CCAN. CENP-A directly recruits CENP-C and CENP-L/N to the centromere (25,26,29,32,43,44). Both CENP-C and CENP-L/N are required for kinetochore recruitment of CENP-/H//I/K/M (32,43). CENP-C directly interacts with CENP-H/K, not CENP-I (32). How CENP-L/N interacts with CENP-H/I/K/M remains unclear. CENP-H/K form a heterodimer through both N-termini and C-termini, and the C-termini of CENP-H/K heterodimer binds to N-terminus of CENP-I. CENP-M directly interacts with the C-terminus of CENP-I and integrate into the CENP-H/I/K/M complex (39).
Fig 3: CENPI depletion inhibits BCa cell proliferation and induces cell cycle arrest and apoptosis. (A) Knockdown efficiency of CENPI in BCa cells was verified by qRT-PCR and WB. β-actin and GAPDH were utilized as loading control for qRT-PCR and WB, respectively. (B) CCK-8 assay was conducted to assess the impact of CENPI depletion on the proliferation of BCa cells. (C) Colony formation assay was performed to evaluate the effect of CENPI depletion on colony-forming ability of BCa cells. (D, E) CENPI depletion induces cell cycle arrest and apoptosis of BCa cells. Flow cytometry analyses were carried out to investigate the effect of CENPI depletion on cell cycle progression and apoptosis of BCa cells. For statistical analyses, three biological replicates were carried out and the results were quantified and represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, One-way analysis of variance (ANOVA)
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