EMBO/soda-nel huggingface dataset:
Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages (2020)
Figure 1

Figure 1. Glufosinate specifically inhibits human recombinant GS

(A) Expression in E. coli and purification of human GS (hGS). Protein was separated by SDS-PAGE and stained with Coomassie Blue dye. M: marker Precision Plus Protein Dual Color Standard (Biorad). From right Lanes 1-4: E. coli Bl21(DE3) cells containing the expression vector without (lanes 1 and 2) and with (lanes 3 and 4) the coding sequence of hGS. Samples were taken immediately before (lanes 1 and 3) and 3,5 h later (lanes 2 and 4) the induction of expression with isopropil-β-D-1-tiogalattopiranoside (IPTG) 0.7mM. The same number of bacteria was analyzed in each sample. Lane 5: isolated and purified inclusion bodies (4 μg). Adiacent boxed lane: Western Blotting analysis of GS.

(B-E) Kinetic study of the reaction catalyzed by recombinant hGS. The reaction, started by adding ATP, was linear for at least 10 min at 25°C (B). Lineweaver-Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence (), or in the presence of glufosinate (C) or MSO (D). Symbols- (): 2 mM MSO or 0.025 glufosinate; () 3 mM of MSO or 0.050 mM of glufosinate; () 4 mM of MSO or 0.065 mM of glufosinate; () 5 mM of MSO or 0.075 mM of glufosinate. The insets represent the secondary plot of the slopes of Lineweaver-Burk plot obtained at the indicated concentrations of glufosinate (C) or MSO (D) used for determining the inhibitor constant Ki. GS inhibition (%) in presence of increasing concentrations of glufosinate () or MSO () results from the average of at least three independent experiments (E). The control value for uninhibited hGS activity is 0.51 ±0.08 μmol/(min x mg protein).

(F-K) Comparative analysis of GS structures from several organisms. Lateral (F) and top view (G) of the entire GS decameric (a dimer of pentamers) structure are reported in pink cartoon representation. (H) Cartoon representation showing the top view of the monomer-monomer interface hosting cofactors, substrates or inhibitors (Dataset EV1) participating to or inhibiting the conversion of glutamate to glutamine. In particular ADP (cyan), Phosphate ions (brown), MSO phosphate (P3S, yellow), phosphoaminiphosphonic acid-adenylate ester (ANP, white), glutamate (magenta), citrate (black) and the imidazopyridine inhibitor ((4-(6-bromo-3-(butylamino)imidazol(1,2-a)pyridin-2-yl)phenoxy) acetic acid, green) are reported in sticks representation, whereas Mn2+ ions are reported in pale blue spheres and Mg2+ in pale green spheres (involved in the coordination of the imidazopyridine inhibitor). (I) Superimposition of all the sampled 17 crystallized structures (Dataset EV1). (J) 2D representation of glutamate, P3S, glufosinate and glyphosate. (K) Zoomed view of the crystallized P3S binding region and the docked glufosinate and glutamate binding regions within the superimposed human (white cartoon) and Z.mays (pink cartoon) GS structures.
Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages (2020)
Figure 2

Figure 2. Glufosinate promotes a rewiring of IL10 macrophages toward a M1-like phenotype through HIF1α stabilization and abolishes immunosuppressive effect of hypoxia

(A-D) Evaluation of M1 markers in macrophages by real-time PCR. Fold change of TNFA, CD80, CXCL9, and CXCL10 mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3).

(E-H) Evaluation of M2 markers in macrophages by real-time PCR. Fold change of MRC1, MSR1, CCL17 and CCL18 mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3).

(I-M) Evaluation of M1 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of TNFA, CXCL10, CD86, CD80 and CXCL9 mRNA in IL10 alone or glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3).

(N-P) Evaluation of M2 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of MRC1, MSR1, and CCL18 mRNA in IL10, glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3).

(Q) Quantification of cancer cell motility through a matrigel coated membrane in presence of IL10, MSO/IL10 and glufosinate (10 and 20µM)- IL10 treated macrophages after 24 h of incubation (n=6).

(R) Evaluation of the capillary network formation in presence of macrophages pretreated for 24 h with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 after 4 h of incubation with HUVEC cells (n=6).

(S) CD8+ T cell suppression by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 for 24 h (n=4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi / well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control.

(T) CD8+ T cell recruitment in a transwell system by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 for 24 h versus macrophages treated with LPS/IFNγ; the migration of T cells cultured without macrophages (Mφ-) in presence of CXCL10 was used as positive control (n=4).

(U) Representative image of Western blotting analysis of HIF1α, REDD1, 4E-BP1, S6 and P70S6K (in their phosphorylated and unphosphorilated form) to test mTOR activation in normoxic (NRX) and hypoxic (HYP) IL10 macrophages treated with glufosinate (20µM), rapamycin (20nM) and a combination of both (n=3).

(V) CD8+ T cell suppression by normoxic and hypoxic IL10 macrophages treated with glufosinate (20µM), rapamycin and a combination of both for 24 h (n=4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi / well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control.

Data are reported as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 **** p < 0.0001.

Exact p values and statistical tests are reported for each experiment in Appendix Table S2.
Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages (2020)
Figure 3

Figure 3. Glufosinate treatment induces an M1-like phenotype and inhibits metastasis.

(A) Experimental plan. Mice were subcutaneously injected with LLC cells. When the tumor reached 80-100mm3 size, mice were treated by gavage with glufosinate (10, 20 and 40 mg/kg) for 14 days.

(B, C) Quantification of glufosinate (B) and glutamine (GLN) (C) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS).

(D, E) Subcutaneous LLC tumor growth over the time (D) and number of lung metastatic nodules in vehicle and glufosinate (10 and 20mg/kg) treated mice (E) (pool of 2 independent experiments; 10 mice per condition in total).

(F-H) Quantification and representative images of F4/80+ CD11c+ cells in the lung metastasis (F), F4/80+ CD206+ (G) and F4/80 CD11c+ (H) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6).

(I, J) RT-PCR quantification of M2 (Ccl22, Arg1 and Ccl17) (I) and M1 (Tnfa, Cxcl9, Nos2, Cd86 and Cd80) (J) markers in vehicle and glufosinate treated mice (n=4).

In each histological quantification n represents the number of animals. Six images per tumor were analysed. Scale bars: 20 µm (F), 50 µm (G-H).

Data are reported as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 **** p < 0.0001.

Exact p values and statistical tests are reported for each experiment in Appendix Table S2.
Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages (2020)
Figure 4

Figure 4. Glufosinate treatment induces CTL accumulation and tumor vessel normalization.

(A-C) Quantification of total length of vessels (A), of CD31+ tumor vessel area (B) and of αSMA+ pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6).

(D) Quantification and representative images of Lectin+ tumor vessel area over the total number of CD31+ vessels in vehicle and glufosinate (20mg/kg) treated mice (n=6).

(E) Quantification and representative images of PIMO+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6).

(F-H) FACS quantification of TCRbeta+ CD8+ cytotoxic T cells in tumors of vehicle and glufosinate-treated mice (F). Evaluation of CD69 (G) and CD25 (H) proteins, expressed as Mean Fluorescence Intensity (MFI), was determined by flow cytometry on the surface of responders CD8+ T cells (n=4).

(I) Quantification of secreted IFNγ in interstitial tumoral fluid of vehicle and glufosinate-treated mice (n=6).

In each histological quantification n represents the number of animals. Six images per tumor were analysed. Scale bars: 50 µm.

Data are reported as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 **** p < 0.0001.

Exact p values and statistical tests are reported for each experiment in Appendix Table S2.
Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages (2020)
Figure 5

Figure 5. Glufosinate impairs metastatic growth after LLC tumor resection

(A) Experimental plan. Mice were subcutaneously injected with LLC cells. When the tumor reached 300-400 mm3 size, mice were tumor resected. 14 days after resection mice were treated by gavage with glufosinate (20mg/kg) for 14 days.

(B) Evaluation of body weight in vehicle and glufosinate (20mg/kg) treated mice (n=8).

(C) Representative images of metastasis at the beginning and end of therapy. Evaluation of the metastatic burden following glufosinate treatment by H&E staining (n=8).

(D) Evaluation of lung total metastatic area and of lung metastatic lesions per size by H&E staining in vehicle and glufosinate (20mg/kg) treated mice (n=8).

(E) Experimental plan for macrophage depletion setting. Mice were preconditioned for 3 days by i.p. injection with anti-CSF1R antibody (or isotype IgG control) before LLC (CD90.1+) tumor cell injection and then injected 3 times a week until sacrifice. When the tumor reached 80-100 mm3 mice were treated by gavage with vehicle or glufosinate (20mg/kg) for 14 days.

(F, G) Quantification (F) and representative images (G) of total metastatic area in LLC-tumor bearing mice preconditioned with IgG or anti-CSF1R antibody and treated with vehicle or glufosinate (20mg/Kg).

In each histological quantification n represents the number of animals. Six images per lung were analysed. Scale bars: 2mm.

Data are reported as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 **** p < 0.0001.

Exact p values and statistical tests are reported for each experiment in Appendix Table S2.
Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages (2020)
Figure 6

Figure 6. Glufosinate treatment promotes M1-like TAMs, CTL tumor accumulation, vessel pruning and normalization, and inhibits metastasis in a murine model of melanoma

(A) Experimental plan. Mice were intradermally injected with YUMM 1.7 (CD90.1+) cells. When the tumor reached 80-100mm3 size, mice were treated by gavage with glufosinate (20mg/kg) or vehicle for 14 days.

(B, C) Intradermal YUMM 1.7 (CD90.1+) tumor growth over the time (B), and number of metastatic lesions per lung (C) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).

(D) Representative images of metastatic lesions checked by DAB-H stain for CD90.1 positivity in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per lung were analysed. Scale bar: 2mm. Zoomed area scale bar: 50µm.

(E, F) FACS quantification of M1-like MHC class IIhigh (E) and MHC class IIlow (F) CD11c+ CD206- TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).

(G) Quantification and representative images of CD11c+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.

(H, I) FACS quantification of M2-like MHC class IIhigh (H) and MHC class IIlow (I) CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).

(J) Quantification and representative images of CD206+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.

(K, L) FACS quantification of M1-like MHC class IIhigh (K) and M2-like MHC class IIlow (L) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).

(M) Quantification of MHC-II protein, expressed as Mean Fluorescence Intensity (MFI), on F4/80+ cells, was obtained by flow cytometry (n=6).

(N) RT-PCR quantification of M2 (Arg1 and Cxcr4) and M1 (Cxcl9 and Nos2) markers in vehicle and glufosinate (20mg/kg) treated mice (n=6).

Data are reported as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 **** p < 0.0001.

Exact p values and statistical tests are reported for each experiment in Appendix Table S2.
Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages (2020)
Figure 7

Figure 7. Glufosinate treatment promotes CTL tumor accumulation, vessel pruning and normalization in a murine model of melanoma

(A-C) Quantification of total length of vessels (A), of CD31+ tumor vessel area (B) and of αSMA+pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.

(D) Quantification and representative images of CA9+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.

(E, F) FACS quantification of CD8+ cytotoxic T cells (E) and CD69+ CD8+ T cells (F) over the total number of CD45+ in vehicle or glufosinate (20mg/kg) treated mice (n=6).

Data are reported as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 **** p < 0.0001.

Exact p values and statistical tests are reported for each experiment in Appendix Table S2.
Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages (2020)
Figure 8

Figure 8. Glufosinate treatment inhibits metastasis in a murine model of breast cancer

(A) Experimental plan. Mice were injected with 4T1 cells in the mammary fat pad. When the tumor reached 80-100mm3 size, mice were treated by gavage with glufosinate (10 mg/kg) or vehicle for 14 days.

(B-D) Evaluation of 4T1 tumor growth over the time (B), mice body weight (C) and number of metastatic nodules (D), in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).

(E-G) Total metastatic area (E) and number of lung metastatic lesions per size by H&E staining (F) with representative images (G) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm.

(H, I) Quantification of F4/80+ macrophage density (H), of F4/80+ CD206+ cells infiltration in the lung metastasis and representative images (I), in vehicle and glufosinate-treated mice (n=6). Six images per lung were analysed. Scale bar: 20µm.

(J) Experimental plan for intravasation setting. Mice were injected with 4T1-GFP+ cells in the mammary fat pad and immediately treated by gavage with glufosinate (10mg/kg) or vehicle for 14 days (neoadjuvant therapeutic regimen). After 14 days of treatment, at the time of tumor resection, blood was collected and analyzed for GFP+ circulating tumor cells (CTCs). Survival studies were performed until the natural death of the post-surgical mice.

(K-M) GFP+ CTCs amount in blood, expressed as GFP expression levels, was determined by qPCR (n=8) (K). Blood was drawn from treated and untreated tumor-bearing mice at the time of tumor resection and plated in culture dishes. The formation of tumor cell colonies was traced over time. Graph shows the number of GFP+ tumor cell colonies (n=8) (L). After treatment and tumor resection overall survival studies were performed until the natural death of the post-surgical mice (n=11) (M).

(N) Experimental plan for extravasation setting. GFP-expressing 4T1 were injected intravenously into Balb/c mice pre-conditioned with glufosinate (10mg/kg) or vehicle for 2 days. After 2 days of treatment, the overall lung metastatic breast cancer cell burden was determined.

(O) GFP+ cancer cells amount in lungs, expressed as GFP expression levels, was determined by qPCR in vehicle and glufosinate (10mg/kg) treated mice (n=8). The results were normalized to the vehicle group.

Data are reported as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 **** p < 0.0001.

Exact p values and statistical tests are reported for each experiment in Appendix Table S2.
Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages (2020)
Figure 9

Figure 9. Glufosinate treatment does not display blood, liver and neuronal toxicity.

(A) Time course evaluation of murine body weight during treatment in vehicle and glufosinate (10 and 20 mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).

(B) Blood counts of WBC and RBC in vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).

(C, D) Evaluation of liver toxicity following glufosinate treatment. Quantification of liver glufosinate (C) and of hepatotoxicity markers ALT (Alanine Aminotransferase) and AST (Aspartate aminotransferase) (D) in serum of vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).

(E-J) Evaluation of neuronal toxicity following glufosinate treatment. Quantification of brain glufosinate (E), of the grip strength for front paws (triangular bar) (F), for all paws (rectangle grid) (G), of the latency to fall off an accelerating rotarod (H), and of the mean paw withdrawal force that caused animals' response in the von Frey test (left hindpaw, I; right hindpaw, J) as an indicator of the development of tactile allodynia in vehicle and glufosinate-treated mice (n=4), before the treatment (baseline), after 1 day (acute Toxicity) and after 15 days (chronic toxicity) (pool of 2 independent experiments; 10 mice per condition in total).

Data are reported as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 **** p < 0.0001.

Exact p values and statistical tests are reported for each experiment in Appendix Table S2.

Legends to Expanded View Figures
Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C (2020)
Figure 1

Figure 1. CENP-A nucleosome binding of the C-terminal fragment of CENP-C

A) Schematic diagram of the functional regions of chicken and human CENP-C molecules. The canonical CENPC motifs for CENP-A binding (aa 655-675 in chicken CNEP-C, aa 738-758 in human CENP-C) are colored pink. Another CENP-A binding region (central domain) in human CENP-C is colored light pink. The C-terminal fragment (aa 601-864: CENPC-CT) and the CENPC motif-containing peptide (aa 643-683; CM peptide) derived from chicken CNEP-C, which were used for the in vitro CENP-A nucleosome binding assay, are diagrammed.

B) Electrophoretic mobility shift assay (EMSA) examination of the binding affinity of CENPC-CT and CM peptide to a CENP-A nucleosome. Binding to a canonical H3 nucleosome was also examined using EMSA.

C) Cryo-EM density map of the CENP-A nucleosome in complex with CENPC-CT (CA-CCCT complex) at a 4.5 Å resolution. The side views of the CA-CCCT complex along the two-fold axis are shown. The density corresponding to each molecule in the complex is color-coded, as indicated in the figure.

D) Cryo-EM structure of the CENP-A nucleosome binding region of CENPC-CT. The left panel shows the cartoon representation of the structure of CENPC-CT bound to the CENP-A nucleosome with the cryo-EM density map. The densities derived from CENPC-CT and CENP-A molecules are color-coded in magenta and pale green, respectively. The functional elements in the CENP-A nucleosome binding region of CENPC-CT are depicted in the right panel. In addition to the CENPC motif, which recognizes the H2A/H2B acidic patch and the CENP-A C-terminal tail (C-tail), the CM upstream and CM downstream regions are identified in the CENP-A bound structure of CENPC-CT. See also Fig. EV2C.
Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C (2020)
Figure 2

Figure 2. RG loopCENP-A recognition through the CM downstream region of CENP-C

A) The RG loopCENP-A binding site of CENPC-CT is shown. The backbone structure of the CENPC-CT is presented in a cartoon model, which is colored in magenta, except for the pink canonical CENPC motif (aa 655-675). The cryo-EM density map of CENPC-CT is overlaid on the cartoon model. The CM downstream region beyond Y675CENP-C is indicated by a dotted box. Key residues involved in binding to the C-terminal tail of CENP-A are indicated as stick models. The cartoon models of CENP-A and histone H4 are shown in pale green and pale blue, respectively.

B) Alignment of sequences around the CENPC motif region of CENP-C from various species: Gg, chicken; Hs, human; Mm, mouse; and Xl, frog. The CENPC motif and the CM downstream region are depicted by pink and purple boxes, respectively, in the sequence alignment. Schematic diagram of chicken CENPC-CT wild-type (CENPC-CT WT), CENPC-CT, in which PSG residues in the CM downstream region were substituted with AAA (CENPC-CT 3A679-681), and CENPC-CT, in which five residues were deleted (CENPC-CT ∆678-683), are shown.

C) EMSA results of the binding affinities of CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 to the CENP-A nucleosome.

D) Stable expression of GFP-fused CENPC-CT and its mutants (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control. Parental CENP-C knockout cells (parental) were also examined.

E) Localization analysis of GFP-fused CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 (green) on the mitotic chromosomes in chicken DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar indicates 10 μm.

F) Localization of CENP-C in CENP-A knockout DT40 cells stably expressing GFP-fused gCENP-A WT, RG-AA, or ∆RG. Scale bar indicates 10 μm. CENP-C was stained by an anti-CENP-C antibody (red) and DNA was stained by DAPI (blue). CENP-C signals on kinetochores in mitotic cells were quantified in each cell. Bar graph indicates mean ± SD (n = 7; ****, P < 0.0001, unpaired t test, two tailed).
Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C (2020)
Figure 3

Figure 3. The CM upstream region of CENP-C stabilizes the CENP-A nucleosome binding

A) Schematic representation of the structure of the CENP-A nucleosome binding region in CENPC-CT. The canonical CENPC motif segment is shown in pink. The CDK1 phosphorylation site (T651) is also indicated. The CM upstream and downstream regions are shown in magenta. Dot circles indicate the recognition sites for H2A/B acidic patch, CENP-A C-terminal tail, and the RG loop in the CENP-A nucleosome.

B) Schematic diagrams of deletion mutants of chicken CENPC-CT. The fragments containing the canonical CENPC motif and the CM downstream region. CENPC643-864, CENPC643-740, CENPC643-740, and CENPC643-683 lack the CM upstream region. CENPC-CT and CENP-C619-689 contain the CM upstream region.

C) EMSA results of the binding affinities of CENP-C fragments indicated in (B) to the CENP-A nucleosome. All fragments were phosphorylated by CDK1.

D) Stable expression of GFP-fused CENPC-CT and CENPC643-864 (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control.

E) Localization analysis of GFP-fused CENPC-CT and CENPC643-864 lacking the CM upstream region (green) on mitotic chromosomes in CENP-C knockout DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.