Functional Effects of AKT3 on Aurora Kinase Inhibitor-induced Aneuploidy*

The suppression of mitotic Aurora kinases (AURKs) by AURK inhibitors frequently causes cytokinetic failure, leading to polyploidy or aneuploidy, indicating the critical role of AURK-mediated phosphorylation during cytokinesis. We demonstrate the deregulated expression of AKT3 in Aurora kinase inhibitor (AURKi)-resistant cells, which we established from human colorectal cancer HCT 116 cells. The AKT family, which includes AKT1, -2, and -3, plays multiple roles in antiapoptotic functions and drug resistance and is involved in cell growth and survival pathways. We found that an AKT inhibitor, AZD5363, showed synergistic effect with an AURKi, VX-680, on two AKT3-expressing AURKi-resistant cell lines, and AKT3 knockdown sensitized cells to VX-680. Consistent with these activities, AKT3 expression suppressed AURKi-induced apoptosis and conferred resistance to AURKi. Thus, AKT3 expression affects cell sensitivity to AURKi. Moreover, we found that AKT3 expression suppressed AURKi-induced aneuploidy, and inversely AKT3 knockdown enhanced it. In addition, partial co-localization of AKT3 with AURKB was observed during anaphase. Overall, this study suggests that AKT3 could repress the antiproliferative effects of AURKi, with a novel activity particularly suppressing the aneuploidy induction.

The dysregulation of cell division causes chromosomal abnormalities in daughter cells, including chromosomal instability and aneuploidy, which are commonly observed in many types of cancer (1). Various mitotic protein kinases, including cyclin-dependent kinases, Aurora kinases (AURKA, 2 -B, and -C), and Polo-like kinases (PLK1-4), are frequently up-regulated in human cancers (2). The mitotic protein kinases are thus considered potential targets for anticancer drugs.
The AURKs, especially AURKA and AURKB, are important in both normal and cancerous cell division (3). AURKA increases in late G 2 /M phase, is mainly located at the spindle poles, and functions in centrosome duplication and mitotic spindle formation (4). Its overexpression is associated with chromosomal instability and rodent fibroblast transformation (5). By contrast, AURKB is located at the centromere until metaphase and then moves to the midzone and midbody until the end of cytokinesis (3). Small molecule AURK inhibitors (AURKis) have been developed, and these cause polyploidy, arising from the failure of cytokinesis (6). VX-680 (Tozasertib, developed by Vertex Pharmaceuticals and Merck), the first clinically tested AURKi, inhibits all three AURKs (7), whereas AZD1152-HQPA (Barasertib, developed by AstraZeneca) and MLN8237 (Alisertib, developed by Millennium Pharmaceuticals and Takeda Pharmaceutical Co.) are highly selective inhibitors of AURKB (8) and AURKA (9), respectively. Their antiproliferative effects are thought to be mediated by mitotic catastrophe, including impaired chromosome segregation, abrogation of the mitotic checkpoint, and cytokinesis failure, causing polyploidy and cell death (10). Molecular predictors of the responses to AURKis would greatly facilitate favorable therapeutic outcomes (11). Previous studies have suggested that the deregulation of TP53, MYC, or drug efflux transporters, known to be involved in resistance to various anticancer drugs, also affects chemosensitivity to AURKis (11). However, the resistance mechanism specific to the antimitotic activity of AURKis is unclear.
In this study, we established AURKi-resistant cells from the human colorectal cancer cell line HCT 116 with VX-680 selection. AKT3 was elevated in several VX-680-resistant clones. AKT was originally discovered as an oncogene in the murine leukemia virus AKT8 (12), and AKT3 is one of the three isoforms (AKT1-3) of the AKT family (13). Phosphoinositide 3-kinase (PI3K)-induced phosphatidylinositol (3)(4)(5) trisphosphate activates AKT through its N terminus pleckstrin homology domain, and the PI3K-AKT axis is a frequently deregulated signal in human cancers (13,14). AKT has antiapoptotic activities and is also involved in resistance to various cytotoxic, antihormonal, and targeted drugs by phosphorylating several apoptosis-associated molecules, including mouse double minute 2 homolog (MDM2), glycogen synthase kinase 3␤ (GSK3␤), Forkhead box subgroup O transcription factor (FOXO), BCL-2-associated death promoter (BAD), caspase-9, p27, and tuberous sclerosis complex 2 (15). Interestingly, previous studies have demonstrated a pharmacological synergy between AKT inhibitors and antimitotic microtubule inhibitors and suggested further study to elucidate the mechanism of the synergy between AKT inhibitors and anti-microtubule drugs for cancer chemotherapy (16 -19). Because AKT3 was elevated in our AURKi-resistant clones, and AKT is activated in G 2 /M phase and promotes G 2 /M progression (20,21), we inferred that AKT signaling affects the antimitotic effects of AURKis. Our data indicate that AKT3 is associated with an AURKi-resistant phenotype and suppresses both apoptosis and aneuploidy.
To examine the drug resistance, we determined the expression of the drug efflux pump ABC transporters by Western blot analysis. An apparent increase of P-glycoprotein (P-GP)/ FIGURE 1. VX-680-resistant clones from HCT 116 cells. A, VX-680-resistant cells were isolated from two independent experiments (origins 1 and 2). In each case, HCT 116 cells were treated with the pan-Aurora kinase inhibitor, VX-680, at the indicated concentrations for several months. The surviving cells were then cloned. B, cellular morphologies of VX-680-resistant clones after drug treatment. Cells were treated with AURKi (100 nM) for 2 days and photographed with a conventional phase-contrast microscope equipped with a fluorescence digital CCD camera (Keyence, BZ-9000, Japan). Representative results are shown. Bar, 100 m. C, cell growth inhibition was examined with WST assays after the cells were treated for 4 days. Open symbols, parental HCT 116 cells; filled and gray symbols, VX-680-resistant cells (116/VX1-1, 116/VX1-2, 116/VX0-1, 116/VX0-2, and 116/VX0-4). Results are presented as means Ϯ S.D. (error bars) from three independent experiments. ABCB1 expression was detected only in the VX0-2 clone, but little expression was seen in other clones; BCRP/ABCG2 was not detected in any of the VX-resistant clones (Fig. 3A). In a cell growth inhibition assay, the VX-resistant clones showed weak resistance to vincristine, an antimitotic microtubule inhibitor and a typical substrate for P-GP/ABCB1, and little resistance to a topoisomerase I inhibitor, SN-38, a typical substrate for BCRP/ABCG2. P-PG/ABCB1-overexpressing HCT 116/MDR cells and BCRP/ABCG2-overexpressing HCT 116/BCRP cells showed strong resistance to these typical substrate drugs, respectively ( Fig. 3B and Table 1). Consistent with the literature, P-GP/ABCB1 and BCRP/ABCG2 conferred very strong resistance to VX-680 and AZD1152-HQPA (22) but did not confer resistance to MLN8237 (Fig. 3C, right graph). By contrast, all VX-resistant clones showed cross-resistance to the three AURKis (Fig. 3D, top graphs).
We next tested the effect of MS-209 (dofequidar), a P-GP inhibitor (23,24), in cell growth inhibition assays. Although MS-209 completely eliminated resistance to VX-680 in HCT 116/MDR cells and AURKi resistance in VX-resistant clones was partly reduced, resistance to the three AURKis was still observed in all VX-resistant clones in the presence of MS-209 ( Fig. 3D and Table  2). Therefore, we speculated that P-GP contributed to the resistance to VX-680 and AZD1152-HQPA in the five VX-resistant clones, but an undetermined factor(s) may have an impact on the cross-resistant phenotype of the VX-resistant clones.   FEBRUARY 3, 2017 • VOLUME 292 • NUMBER 5 AURKB phosphorylates mitotic histone H3 at Ser-10 (25). Thus, we examined the inhibitory effect of VX-680 on histone H3 phosphorylation at Ser-10 (P-H3S10) in mitotic VX-resistant clones by immunofluorescence confocal microscopic analysis (Fig. 4, A and B, and Table 3). VX-680 treatment severely inhibited the P-H3S10 signal in parental HCT 116 cells but not in HCT 116/MDR cells (Fig. 4A, left panels), suggesting that P-GP-mediated efflux of VX-680 from cells protected AURKB activity. However, VX-680 treatment reduced P-H3S10 in all VX-resistant clones except VX1-2 ( Fig. 4, A and B), indicating that AURKB was inhibited by VX-680 in VX0-1, 0-2, 0-4, and 1-1 clones. Because P-H3S10 was not suppressed by VX-680 in the P-GP-negative VX1-2 clone, AURKB activity seemed to be unaffected in the VX1-2 clone in the presence of VX-680. We analyzed the genomic DNA sequence of AURKB in the VX1-2 clone and found a point mutation causing an amino acid substitution at position 250, H250Y (Fig. 4B). The same mutation H250Y of AURKB was reported to cause marginal resistance to VX-680 by hyperactivating the catalytic activity of the kinase (26). Thus, we speculated that the AURKi resistance of VX1-2 would be mediated by the H250Y mutation in AURKB.

AKT3 Represses Aneuploidy
AURKis exert antiproliferative activities through inducing both cell death and polyploidy (7,27). Therefore, we next investigated the expression of apoptosis-related molecules by Western blot analysis in cells treated with AURKis (Fig. 4C). The expression of AURKA, AURKB, and proapoptotic BAX was quite similar in the VX-resistant cells and parental cells. Although P21 and TP53 were induced by AZD1152-HQPA in clones VX0-4, 1-1, and 1-2, their expressions were reduced by VX-680 and MLN8237 in all VX-resistant clones. Importantly, cleaved caspase-9, an initiator of the mitochondria-related intrinsic apoptosis pathway (28), induced by the three AURKis (clearly induced by 100 nM AURKi), was suppressed in all VXresistant clones. This indicates that the caspase-9-initiated apoptosis induced by AURKis was suppressed in the VX-resistant clones.
Deregulation of AKT3 Suppresses AURKi-mediated Apoptosis and Aneuploidy-Our Western blot analysis showed that AKT3 was overexpressed in four of five resistant clones, but the other AKT family members, AKT1 and AKT2, were not (Fig.  5A). AKT phosphorylation (Ser-473) was increased in the VXresistant cells, and GLUT1 and GSK-3␤ phosphorylation at Ser-9, which are linked to AKT signaling, were increased ( Fig.  5A), suggesting activation of the AKT pathway. GLUT1 is reportedly involved in cytokinesis (29), and our data suggest a

AKT3 Represses Aneuploidy
possible role for AKT3 in the AURKi-resistant phenotype.
Although growth inhibition assays showed little resistance to the AKT inhibitor AZD5363 in these VX-resistant clones (Fig.  5, B and C), the addition of AZD5363 after treatment with VX-680 sensitized the VX-resistant cells, especially VX0-1 and VX0-4, to VX-680 (Fig. 5D). Sensitivity of parental HCT 116 to VX-680 cells appeared to be unaffected by the AKT inhibitor, but calculated combination index values were Ͻ1 in VX-resist-ant clones, especially in VX0-1 and VX0-4 clones (Fig. 5D). Therefore, there may be a selective synergistic effect of the AKT inhibitor on VX-680 resistance (30).
To examine the effect of AKT3 on AURKi sensitivity, AKT3 knockdown experiments were performed in VX0-1 and VX0-4 clones (Fig. 5, E and F). AKT3 knockdown efficiency was transient and incomplete in VX0-1 cells, but significant sensitization to VX-680 and MLN8237 was observed (Fig. 5E). The

. Effect of AURKi on the VX-resistant cells.
A and B, mitotic phosphorylation of histone H3 in control and VX-680-treated (100 nM for 10 h) cells after drug treatment. Mitotic phosphorylation of histone H3 (P-H3S10; green signal) was probed with anti-phosphohistone H3 (Ser-10) antibody and Alexa Fluor 488-conjugated secondary antibody. Blue signal indicates DNA counterstained with DAPI and visualized with confocal microscopy. Representative images are shown. Cells containing mitotic chromosomes (Ͼ50 cells/sample) were analyzed, and the ratio of the green signal (phospho-H3) was calculated (summarized in Table 3). Mutation of AURKB genomic DNA in VX1-2 cells is shown by a DNA sequencing chromatogram in B. C, Western blotting analysis of the expression of apoptosis-related molecules in VX-680-resistant cells after drug treatment at the indicated concentrations for 2 days. Whole cell lysates were prepared, and the induction of cleaved caspase-9, TP53, and P21 was attenuated in the resistant cells. Representative results are shown. transfection efficiency of siRNA transfection in VX0-4 cells was poor; thus, we performed AKT3 knockdown by shRNA plasmid transfection and found significant sensitization to VX-680 and MLN8237 in the VX0-4 clone (Fig. 5F). These data indicate that the AKT3 pathway has the ability to contribute to chemoresistance against AURKis.
We also tested the impact of AKT3 on the pharmacological effects of AURKis in HeLa cells. Active AKT3, either myr-AKT3 (31) or AKT3 (E17K) (32), was transiently expressed in HeLa cells, and apoptosis and aneuploidy caused by AZD1152-HQPA were analyzed with flow cytometry (Fig. 7, A and B). The results showed that the expression of active AKT3 and GLUT1 suppressed both the apoptosis and aneuploidy caused by AZD1152-HQPA in HeLa cells (Fig. 5B). AKT3 also suppressed the apoptosis induced by two other AURKis (data not shown). Cell growth inhibition assays showed that IC 50 values against VX-680 were increased by AKT3 expression, suggesting that AKT3 conferred resistance to VX-680 in HeLa cells (Fig. 7C).
Because the aneuploidy-suppressive effect of AKT3 was a novel activity, we further examined anti-aneuploidy activity of AKT3 by analyzing nuclear size in aneuploidy/polyploidy cells by confocal microscopy. In addition to HCT 116 cells, we also tested the effect of AKT3 on MCF7 and OVCAR3 cell lines, because these cells do not express endogenous AKT3 (supplemental Fig. S1). The nuclei were stained with 4Ј,6-diamidino-2-phenylindole (DAPI), and AKT3-transfected cells were recognized by staining with an anti-hemagglutinin (HA) antibody (Fig. 7D). Consistent with FACS analysis ( Fig. 2A), nuclear sizes of AZD1152-HQPA-treated cells were bigger than those of nocodazole-treated cells arrested at G 2 phase (Fig.  7D and supplemental Fig. S2), suggesting that the nuclear size of polyploidy cells was bigger than that of G 2 phase cells. The sizes of nuclei were measured in captured images with the ImageJ software, and the data were summarized as a box plot with a bee swarm dot plot overlay (Fig. 7E). AZD1152-HQPA increased the median nuclear size with a range of sizes in the control cells by the induction of aneuploidy, whereas active AKT3 expression repressed this increase of nuclear size after ADZ1152-HQPA treatment. Anti-aneuploidy activity of AKT3 was also observed in myr-AKT3-stably expressing HCT 116 cells (supplemental Fig. S3).
Ovarian cancer OVCAR5 cells express AKT3 and showed relative resistance to VX-680 compared with OVCAR3 cells, which do not express AKT3 (supplemental Fig. S1). We next tested the effect of AKT3 knockdown on AURKi-induced aneuploidy (Fig. 8). As in Fig. 7, we analyzed AURKi-induced nuclear size change in AKT3 siRNA-transfected cells (Fig. 8B). The results showed that the ratio of cells with aneuploid large nuclei was increased in AKT3-reduced cells after VX-680 treatment but not after nocodazole treatment (Fig. 8, C and D). Collectively, our data indicate that AKT3 can repress the induction of aneuploidy by AURKi.
Localization of AKT3 and AURKB during Anaphase-Some GLUT1 localizes to the midbody and is involved in the pro-  FEBRUARY 3, 2017 • VOLUME 292 • NUMBER 5 gression of cytokinesis (29). Consistent with this, we found that the location of GLUT1 partly overlapped with that of AURKB in the midbody in AKT3-expressing MDA MB-231 cells (supplemental Fig. S1). We further investigated the subcellular localization of AKT1 (Fig. 9, A and D), AKT2 (Fig. 9, B and E), and AKT3 (Fig. 9, C and F) during mitosis in the VX0-1 clone and MDA-MB 231 cells using confocal microscopy (Fig. 9). Most endogenous AKT1, -2, and -3 (red signal) were not on the chromosomes (blue signal) during mitosis. However, during anaphase, we observed partial colocalization of AKT3 (red signal) and AURKB (green signal), especially around the central region of the cell, as detected by merged yellow signals (Fig. 9, C and F). In a three-dimen-sional reconstructed movie, rotated at right angles, yellow signals indicated overlapping of AKT3 and AURKB during anaphase in MDA-MB 231 cells (supplemental Movie S1). A signal intensity profile showed that AKT3 (red signal) was partly coincident with AURKB (green signal) in the central region during anaphase (Fig. 9G). AKT1 and AKT2 were also detected during mitosis, and the locations of AKTs and AURKB also, in part, seemed to overlap during anaphase (Fig. 9, A, B, D, and E). These results suggest that AKT3 and AURKB partly colocalize in the central midzone during anaphase. Collectively, these observations suggest that AKT3 has a role in mitosis and potential to repress AURKi-induced cytokinetic dysregulation.  Table S3. Statistical analysis was performed with the Mann-Whitney U test.

Discussion
AURKA and AURKB are important regulators of mitotic processes, and their deregulated expression has been demonstrated in various cancers (10). Therefore, AURKs are considered good pharmacological targets for anticancer chemotherapeutics, and various AURKis have been developed for therapeutic use (27). During the investigation of AURKi-resistant cells, we identified AKT3 with an ability to suppress AURKi-induced aneuploidy. Our data suggest that AKT3 has a potential to contribute to proper cytokinesis progression.
Previous studies have investigated the AURKi-resistant phenotypes of human cells and have shown that a point mutation in AURKB reduces its sensitivity to its inhibitor (26). Our experiments show that the mitotic P-H3S10, a typical target of AURKB (25), was inhibited by the pan-AURKi VX-680 in four of the five VX-resistant cells, whereas P-H3S10 in VX1-2 clone, which harbors the AURKB mutation H250Y, was not inhibited. Therefore, we concluded that AURKB was inhibited by VX-680 in four of the five resistant clones. Another study showed that P-GP/ABCB1 and BCRP/ABCG2 confer resistance to AZD1152-HQPA (22). Because our VX-resistant cells showed cross-resistance to P-GP-insensitive MLN8237, and the P-GP inhibitor MS-209 did not abolish the resistance of the VX-resistant cells, we presume that P-GP and BCRP contribute only slightly to the AURKi-resistant phenotype of our VX-resistant cells. Because apparent P-GP expression was detected only in the VX0-2 clone, elevations of P-GP and AKT3 were not observed in the VX1-1 clone, and an apparent pharmacological synergy between the AKT inhibitor and VX-680 was seen in only two VX-resistant VX0-1 and 0-4 clones (supplemental Table S1), there is a possibility that multiple different resistance mechanisms might be involved in each VX-resistant clone.
In addition, our VX-resistant clones are near tetraploidy cells, and TP53 accumulation after AURKi treatment was  Table S4. Statistical analysis was performed with the Mann-Whitney U test. reduced in the clones (Fig. 4C), suggesting that the VX-resistant cells should proliferate after VX-680-induced cytokinetic failure. Tetraploidy after cytokinesis failure is reported to activate the hippo pathway and stabilize TP53 in postmitotic G 1 arrest, and conversely growth factor signaling, such as IGF1, is sufficient to overcome such G 1 arrest (33). IGF1 activates the PI3K-AKT pathway, and numerous studies have shown that the activation of the PI3K-AKT pathway is associated with a poor prognosis and the chemotherapeutic resistance of cancer cells (13,14). Although our preliminary microarray gene expression analysis did not indicate an activation of the hippo pathway, 3 we found overexpression of AKT3 in the VX-resistant clones (Fig.  5A). We confirmed that AKT3 suppresses AURKi-induced caspase-9 activation and confers resistance to AURKi, whereas the AURKi-induced accumulation of TP53 and P21 was not abolished by AKT3. A previous study reported that reactive oxygen species (ROS) and p38 MAPK activate P21 in AURKiinduced aneuploid cells (34), and the increased ROS production in aneuploid cells is attributed to high glucose metabolism (35). Intriguingly, the glucose metabolic pathway seems to be altered in our AURKi-resistant clones, because the expressions of the glucose transporter GLUT1 and some glycolysis-regulating enzyme genes were up-regulated in the VX-resistant clones (data not shown). Therefore, the AURKi-resistant clones have potentially high glucose metabolism and ROS production. Because the AURKi-induced accumulation of TP53 and P21 was reduced in our AURKi-resistant clones, the ROS-associated downstream mechanism(s) may be altered. A future study should investigate these phenomena to identify as yet undetermined resistance mechanisms to AURKi.
The AKT pathway is involved in cell cycle progression, and its activity increases during mitosis and promotes the G 2 /M transition (20,21). Recent studies also showed complicated positive-and negative-regulatory networks among AURKA, AURKB, PLK, and AKT during mitosis (36 -40), whereas AKT3 expression conferred slight resistance to the PLK inhibitor in our setting (Fig. 6B). AKT could activate PLK1 (39), and other studies reported that PLK1 could activate PI3K-AKT signaling by suppressing PTEN (phosphatase and tensin homolog) (41,42). This possible cross-talk between AKT3 and PLK1 signalings might be involved in AKT3-induced cross-resistance to PLK inhibitor.
Moreover, in this study, we demonstrated that AKT3 suppresses AURKi-induced aneuploidy in colorectal cancer HCT 116 and other cells. Although the specific role of AKT3 in colorectal cancer has not been established, previous studies have suggested AKT3 as a possible mitotic regulator (43,44). Because the inhibition of AURKB causes cytokinetic failure and aneuploidy, and a possible role of AKT in microtubule stabilization has been suggested (45), we suspected that AKT3 may have non-apoptotic activity associated with the cytokinetic machinery, which might counteract aneuploidy induction by AURKis and modulate the chemosensitivity of cancer cells to AURKi. Undoubtedly, AURKis disturb AURKB-regulated various central spindle-associated factors, such as KIF23, Rac-GAP1, KIF2A, and KIF4A (46 -48), and thus AKT3 might affect various functions of these molecules. Further study is required to uncover the entire mechanism and pathway of AKT3-mediated prevention against aneuploidy by AURKis.
In addition, the expression of AKT3 is observed in several types of cancer and some patients (Cancer Genome Atlas database), so that AURKi should be administered to these patients with care, and the deregulated expression of cytokinetic molecules might affect the therapeutic effects of various antimitotic drugs for cancer.
Plasmids-Human wild-type AKT3 cDNA (NM_005465.4) was cloned with conventional PCR and inserted into the CMV promoter-driven plasmid pD3HA, as described previously (52). The SRC myristoylation signal sequence peptide (MGSSK-SKPK; designated "myr") was fused to the N terminus of AKT3 to construct active AKT3 by conventional PCR (31). AKT3 was expressed with an HA tag at the C terminus. PCR primers used in this study are listed in supplemental Table S1.
Genomic DNA Sequence for AURKB-Genomic DNA from VX-resistant clones was prepared using the DNeasy blood and tissue kit (Qiagen, Valencia, CA), and DNA fragments corresponding to the AURKB open reading frame (genome assembly data: GRCh38) were amplified by PCR and directly sequenced. PCR primers used in this study are listed in supplemental Table S2.
Cells were seeded on Scientific TM Nunc TM Lab-Tek TM II CC2 TM chamber slides (5-10 ϫ 10 4 cells/0.5 ml/well; Thermo Fisher Scientific) and cultured overnight. Cells were then transfected with plasmids using FuGENE HD transfection reagent (Promega) and fixed the next day with ice-cold methanol or 3.7% formalin, phosphate-buffered saline (PBS) for 10 min at room temperature. Cells were permeabilized with NEB100 buffer for 10 min at room temperature, washed with PBS, and blocked with 3% bovine serum albumin (BSA) in TBST (20 mM Tris-HCl (pH 8), 150 mM NaCl, 0.05% Tween 20) for 30 min at room temperature, as described previously (54). The slides were incubated overnight at 4°C with primary antibodies diluted with 3% BSA, TBST and then with Alexa Fluor 488conjugated goat anti-mouse or Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Molecular Probes), diluted 1:2000 in 3% BSA, TBST for 2 h at room temperature. For primary antibodies, we used anti-AKT1 (C73H10), anti-AKT2 (D6G4), anti-AKT3 (E1Z3W) rabbit antibodies diluted at 1:3000. Anti-AURKB mouse monoclonal antibody (ab3609, Abcam) was used at 0.2 g/ml in 3% BSA, TBST. Reaction specificities of anti-AKT antibodies are shown in supplemental Fig. S4. AKT3-HA was detected by anti-HA antibody clone 3F10 or 12CA5 (diluted at 10 or 500 ng/ml, respectively). Anti-phosphohistone H3 (Ser-10) antibody (clone RR002) was purchased from Merck Millipore (Darmstadt, Germany) and incubated with cells for 2 h at room temperature (dilution at 0.5 g/ml). Cells were washed three times with PBS and mounted with Prolong Gold Antifade Reagent with DAPI (Invitrogen). Acquisition of images (640 ϫ 640 pixels) was performed using the FV1000-D IX81 confocal microscope equipped with an objective lens, UPLFLN ϫ40, numerical aperture 1.3 oil (Olympus Corp., Tokyo, Japan) with identical parameter settings. The EGFP signal intensity and region integration profile was analyzed with the associated viewer software (Olympus). Confocal two-dimensional TIFF images were merged with the Adobe Photoshop CS4 Extended software (Adobe Systems Inc., San Jose, CA), and confocal three-dimensional movies were made with the accompanying FV10-ASW software, according to the instruction manual (Olympus Corp.).
Statistical Analysis-In vitro experiments were repeated independently at least twice, and similar results were observed. Quantitative results are presented as means Ϯ S.D. (n Ն 3). A two-tailed Student's t test was used to evaluate the significance of differences between two groups of data with similar variances. A value of p Ͻ 0.05 was considered statistically significant. A box plot and bee swarm dot plot were generated with BoxplotR software (55). The Mann-Whitney U test was used to evaluate the statistical significance of the box plot results, with IBM Statistics SPSS version 22.0 (IBM Corp.).