![Figure 3. Relationship of the EB1/microtubules and the histone phosphorylation pathways in CPC localization. (A) HeLa cells treated with 0.33 and 3.3 µM nocodazole for 7 h were fixed and stained with tubulin and Aurora B antibodies. The inset is a projection of six z sections. White arrowheads in 3.3 µM nocodazole (Noc; tubulin images) point to centrosomes. Bars: (main images) 1.8 µm; (inset) 0.22 µm. Settings that allow visualization of spindle microtubules obscure the microtubule foci in 0.33 µM nocodazole, so we have not shown control cells treated with DMSO. (B) Box and whisker plot of centromeric Aurora B levels measured in HeLa cells after the indicated treatments. The lines within the boxes represent the medians. Mean inner centromeric Aurora B levels from the same experiment shown in Fig. S3 A. *, P = 0.01182. (C) HeLa cells were treated with 10 µM reversine and 1 µM 5-iodotubericidine (HI) separately or in combination (reversine [R] + HI) in the presence of MG132. (Reversine only is shown in Fig. S3 B, and HI only with the same control cells is shown in Fig. S3 C.) To determine whether microtubules can recruit Aurora B in the absence of phosphohistone marks, HeLa cells were treated with or without 3.3 µM nocodazole along with the reversine, HI, and MG132 (reversine + HI + nocodazole). Control cells were treated with MG132 only. Cells were fixed after 30 min and stained with anti-pH3T3, anti-tubulin, and anti–Aurora B antibodies. Bright-field image (reversine + HI + nocodazole treatment) to show that there is a cell that lacks detectable staining. Bar, 1.7 µm. (D) Centromeric Aurora B levels were measured at indicated treatment conditions. In this experiment, 1 µM reversine was used. *, P = 6.65 × 10−127. (E) Expression of CENPB-INCENP fusion protein in U2OS-TR cells rescued the reduction of Bub1, phospho-KNL1, and pH3T3 levels after EB1 depletion. A stable U2OS-TR line was either mock treated (control) or EB1 siRNA treated with or without CENPB-INCENP induction. Bar, 1.8 µm. (F) Quantification of Bub1 levels. *, P = 1.05 × 10−262. (G) Quantification of phospho-KNL1(Ser60) levels. *, P = 8.95 × 10−31. Error bars show standard deviations. The height of the boxes represents the IQR. The central horizontal lines depict the median. The top whiskers represent the 75th percentile + 1.5× IQR, and the bottom whiskers represent the 25th percentile − 1.5× IQR. ACA, anticentromere antigen; a.u., arbitrary units; res, resistant.](https://cdn.rupress.org/rup/content_public/journal/jcb/204/6/10.1083_jcb.201307119/7/jcb_201307119r_fig3.jpeg?Expires=1771625642&Signature=xC4GET2Kyx5MxPnxEMUOEJvb5vj8iA-yWjwUkEwN2A9C~0REbuBzKnINGdd8fCp5eucJpZCexGXoFzG5B4NLjTkc1fDSz6r3ri5ZpFiLPPmGrmZ3Spj~97HjM6uQMavij~BUdGAs8B979KgVpHRC-4fqKzIskNaVUtLuMihFDQQZcIso~fqWa6WgrEmqBm1EnpMniuPZ4vQgJoeyTAg7twIUV2vU~FDLCgF4WZfHCU1oIvbzB3I0hdog9V-Pc5h2h-Ik~IYo1e7L0SRgiHNuYDjCsVeiyXP7fB2cSbzGFYzYI3kMu5etieqBjyPHlF2WOFi55Ekj2L-P~NqhE74osg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Relationship of the EB1/microtubules and the histone phosphorylation pathways in CPC localization. (A) HeLa cells treated with 0.33 and 3.3 µM nocodazole for 7 h were fixed and stained with tubulin and Aurora B antibodies. The inset is a projection of six z sections. White arrowheads in 3.3 µM nocodazole (Noc; tubulin images) point to centrosomes. Bars: (main images) 1.8 µm; (inset) 0.22 µm. Settings that allow visualization of spindle microtubules obscure the microtubule foci in 0.33 µM nocodazole, so we have not shown control cells treated with DMSO. (B) Box and whisker plot of centromeric Aurora B levels measured in HeLa cells after the indicated treatments. The lines within the boxes represent the medians. Mean inner centromeric Aurora B levels from the same experiment shown in Fig. S3 A. *, P = 0.01182. (C) HeLa cells were treated with 10 µM reversine and 1 µM 5-iodotubericidine (HI) separately or in combination (reversine [R] + HI) in the presence of MG132. (Reversine only is shown in Fig. S3 B, and HI only with the same control cells is shown in Fig. S3 C.) To determine whether microtubules can recruit Aurora B in the absence of phosphohistone marks, HeLa cells were treated with or without 3.3 µM nocodazole along with the reversine, HI, and MG132 (reversine + HI + nocodazole). Control cells were treated with MG132 only. Cells were fixed after 30 min and stained with anti-pH3T3, anti-tubulin, and anti–Aurora B antibodies. Bright-field image (reversine + HI + nocodazole treatment) to show that there is a cell that lacks detectable staining. Bar, 1.7 µm. (D) Centromeric Aurora B levels were measured at indicated treatment conditions. In this experiment, 1 µM reversine was used. *, P = 6.65 × 10−127. (E) Expression of CENPB-INCENP fusion protein in U2OS-TR cells rescued the reduction of Bub1, phospho-KNL1, and pH3T3 levels after EB1 depletion. A stable U2OS-TR line was either mock treated (control) or EB1 siRNA treated with or without CENPB-INCENP induction. Bar, 1.8 µm. (F) Quantification of Bub1 levels. *, P = 1.05 × 10−262. (G) Quantification of phospho-KNL1(Ser60) levels. *, P = 8.95 × 10−31. Error bars show standard deviations. The height of the boxes represents the IQR. The central horizontal lines depict the median. The top whiskers represent the 75th percentile + 1.5× IQR, and the bottom whiskers represent the 25th percentile − 1.5× IQR. ACA, anticentromere antigen; a.u., arbitrary units; res, resistant.
