Fig 1: ANP32E promotes GC cell proliferation via NUF2 upregulation. (A) Spearman's correlation between ANP32E and NUF2 expression levels was analyzed in tumor tissues using TCGA. r=0.6, P<0.05. (B) NUF2 expression levels in tumor (n=408) and adjacent normal (n=211) tissues were analyzed using TCGA. (C) Reverse transcription-quantitative PCR was used to determine NUF2 mRNA expression levels in Ctrl and ANP32E-overexpressing HGC27 cells. The NUF2 mRNA expression levels were normalized to GAPDH. (D) Luciferase reporter assay was performed to determine the interaction between ANP32E and NUF2. (E) Western blotting was used to determine NUF2 protein expression levels of NUF2 in Ctrl + siCtrl, ANP32E-overexpressing + siCtrl and ANP32E-overexpressing + siNUF2 HGC27 cells. . (F) Cell proliferation and (G) colony formation was assessed in Ctrl + siCtrl, ANP32E-overexpressing + siCtrl and ANP32E-overexpressing + siNUF2 HGC27 cells. (H) PI/Annexin V-FITC staining and flow cytometry were performed to analyze cell apoptosis in Ctrl + siCtrl, ANP32E-overexpressing + siCtrl and ANP32E-overexpressing + siNUF2-treated HGC27 cells. *P<0.05, **P<0.01 and ***P<0.001. ANP32E, acidic nuclear phosphoprotein 32 family member E; GC, gastric cancer; NUF2, NUF2 component of NDC80 kinetochore complex; TCGA, The Cancer Genome Atlas; si, small interfering RNA; Ctrl, control.
Fig 2: Dsn1 phosphorylation by Aurora B modulates kinetochore disassembly in late anaphase(A) HeLa cells expressing Dsn1-WT-GFP were stained for DNA (blue), GFP (green), Nuf2 (red), and CENP-C (gray). Where indicated, cells were treated with 10 µM ZM447439 for 15 min prior to fixation.(B) HeLa cells expressing Dsn1-EE-GFP were treated and stained as in (A).(C) In anaphase cells treated as in (A) and (B), Nuf2 at individual kinetochores was quantified as a function of distance from the midzone. Dots are shaded as in Figure 1D. Using linear regression, for 17 Dsn1-WT-GFP cells, slope = -2.9 and is non-zero (p < 0.0001, F test). For 17 ZM447439-treated Dsn1-WT-GFP cells, 20 Dsn1-EE-GFP cells, and 16 ZM447439-treated Dsn1-EE-GFP cells, the slopes are -0.3, 0.03, and -0.1, respectively, which are not significantly different from zero (p = 0.29, 0.86, and 0.60). Comparing Nuf2 in Dsn1-WT-GFP cells with and without ZM447439, and comparing Nuf2 in Dsn1-WT-GFP with Dsn1-EE-GFP cells, the slopes are significantly different from one another (p < 0.0001, F test). Confidence intervals (95%) are shown as fine lines.(D) In telophase cells treated as in (A) and (B), Nuf2 was quantified at kinetochores. Colored symbols show the results for individual kinetochores, black dots show the means for individual cells, and red bars show the means of these means. For Dsn1-WT-GFP cells ± ZM447439, n = 9 and 12; for Dsn1-EE-GFP cells ± ZM447439, n = 11 and 7. **Adjusted p = 0.0054; ****adjusted p < 0.0001, by one-way ANOVA followed by Dunnett’s multiple comparisons test.Scale bars: 5 µm (A and B).
Fig 3: Loss of kinetochore attachment at centromeres after dislocation. a Representative images showing the kinetochore complex detaches from the core centromere, whilst remaining connected by PICH- or RPA-coated DNA threads (arrows) in BI2536-treated metaphase-like RPE1 cells. Inner and outer kinetochores were labelled by CENPA and NUF2, respectively. b Examples showing the majority of centromeres retain two kinetochores in pre-collapsed mitotic populations (prometaphase and metaphase-like cells) after BI2536 treatment. c Representative image showing the metaphase-collapse cells losing kinetochore complex at one side of the centromere. Note: the side without the kinetochore is concomitant with the formation of PICH-associated DNA linkages (arrows). Enlarged images (1 and 2) highlight the loss of CENPA signal at regions of where PICH-decorated DNA linkages form (arrows). d Quantification of the numbers of CENPA- and NUF2-labelled kinetochores at centromeres in prometaphase, metaphase-like and collapse stages after BI2536 treatment (n = 3 independent experiments analysing a total of 3020 CENPA-labelled centromeres and 3625 NUF2-labelled centromeres; mean ± S.D. is shown). Note: all RPE1 cells analysed were pre-synchronised at G1/S by single thymidine block. Drugs were added at 6 h post-release. After 2 h treatment, cells were subject to immunofluorescence staining. Scale bars = 5 µm
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