Journal: Cell research

Article Title: Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis.

doi: 10.1038/cr.2008.11

Figure Lengend Snippet: Figure 1 Kinetochore localization of cyclin B1. (A) HeLa cells were immunostained by anti-cyclin B1 antibody. The selected regions were magnified and the kinetochore localization of cyclin B1 was judged by its immunostaining patterns with paired dots on both sides of each chromosome as indicated by arrows. Bar = 5 µm. (B) HeLa cells expressing GFP-cyclin B1 were fixed with methanol and stained with anti-Cenp A antibody to indicate kinetochores. The kinetochore localization of GFP-cyclin B1 is indicated by arrows. Bar = 5 µm. (C) Still images of time lapse experiments with HeLa cells expressing GFP-cyclin B1showing the kinetochore localization of cyclin B1 in prometaphase. The kinetochore localization of cyclin B1 is indicated with arrows. Time is given in minutes. Bar = 5 µm. (D) Chromosomal spreads showing the localization of cyclin B1 on chromosomes and kinetochores from prophase to metaphase. Cenp A (red) was co-immunostained with cyclin B1 (green). Selected regions are magnified, showing the chromosome and kinetochore localization of cyclin B1. Bar = 5 µm.

Article Snippet: Cell extracts and Western blotting After being resolved on 10% SDS-PAGE gels, the protein samples were transferred onto nitrocellulose membrane in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) for 1 h at 100 V. The filters were then blocked in TTBS [20 mM Tris-HCl (pH 7.4), 500 mM NaCl and 0.3% Tween-20] containing 5% non-fat milk overnight at 4 °C and probed with primary antibodies diluted in TTBS containing 5% non-fat milk, monoclonal antibody against cyclin B1 (Upstate) diluted 1:1000, goat polyantibody against cyclin A2 (Santa Cruz) diluted 1:1000, monoclonal antibody against cyclin B2 (Santa Cruz) diluted 1:1000, rabbit serum against GFP (generated by injecting rabbit) diluted 1:5000.

Techniques: Immunostaining, Expressing, Staining

Journal: Cell research

Article Title: Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis.

doi: 10.1038/cr.2008.11

Figure Lengend Snippet: Figure 2 Cyclin B1 is localized on the outer plate of kinetochores. (A) Cyclin B1 co-localized with HEC1 on the outer plate of kinetochores. HeLa cells expressing GFP-cyclin B1 were fixed by methanol and stained with anti-HEC1 antibody. Selected regions are magnified showing the colocalization of cyclin B1 (originally green) and Hec1 (originally red). The kinetochore lo- calization of cyclin B1 and HEC1 is indicated with arrows. Bar = 5 µm. (B) The localization of cyclin B1 compared with Aurora B on kinetochores. HeLa cells expressing GFP-cyclin B1 (originally green) were fixed and stained with anti-Aurora B antibody (originally red). Selected regions are magnified comparing the localization between cyclin B1 and Aurora B. The kinetochore localization of cyclin B1 and the centromere localization of Aurora B are indicated with arrows. (C) HeLa cells co-immunostained by anti-cyclin B1 (originally green) and anti-Plk1 (originally red) antibodies showing their colocalization. Bar = 5 µm.

Article Snippet: Cell extracts and Western blotting After being resolved on 10% SDS-PAGE gels, the protein samples were transferred onto nitrocellulose membrane in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) for 1 h at 100 V. The filters were then blocked in TTBS [20 mM Tris-HCl (pH 7.4), 500 mM NaCl and 0.3% Tween-20] containing 5% non-fat milk overnight at 4 °C and probed with primary antibodies diluted in TTBS containing 5% non-fat milk, monoclonal antibody against cyclin B1 (Upstate) diluted 1:1000, goat polyantibody against cyclin A2 (Santa Cruz) diluted 1:1000, monoclonal antibody against cyclin B2 (Santa Cruz) diluted 1:1000, rabbit serum against GFP (generated by injecting rabbit) diluted 1:5000.

Techniques: Expressing, Staining

Journal: Cell research

Article Title: Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis.

doi: 10.1038/cr.2008.11

Figure Lengend Snippet: Figure 3 Displacement of cyclin B1 from kinetochores by microtubule attachment and the role of the dynein/dynactin complex. (A) When HeLa cells were treated with nocodazole to disassemble microtubules, the accumulation of cyclin B1 at kineto- chores was enhanced. Control (DMSO) and nocodazole-treated cells were fixed and immunostained with a mouse anti-cyclin B1 antibody. The kinetochore localization of cyclin B1 is determined by its immunostaining patterns with paired dots on both sides of each chromosome and is indicated with arrows. The mean intensity of cyclin B1 on kinetochores was shown in both DMSO- and nocodazole-treated cells (n > 100 pairs of kinetochores in 8 cells in each category). Error bars indicate SD. Bar = 5 µm. (B) HeLa cells expressing GFP-cyclin B1 were blocked in prometaphase by nocodazole treatment for 2 h and released for 1 h under the treatment of DMSO (control) or MG132. Cells with several unaligned chromosomes were selected to show the localization of cyclin B1 on the unattached kinetochores in contrast to the microtubule-attached kinetochores. Bub1 was immunostained to show the unattached kinetochores (red). A selected region is magnified to show the kinetochore localization of cyclin B1. Unattached kinetochores are indicated with arrows. Bar = 5 µm. (C) The kinetochore localization of cyclin B1 was regulated by the dynein/dynactin complex. (a) Cells in a control experiment were treated with saline g (Saline). (b) Cells were depleted of ATP by treatment with azide and deoxyglucose (AZ/DOG). (c) Nocodazole pretreated cells were treated with azide and deoxyglucose (Noc pretreated + AZ/DOG). Cells were immunostained with anti-HEC1 antibody to show kinetochores (red). The kinetochore localization is indicated with an arrow. Bar = 5 µm.

Article Snippet: Cell extracts and Western blotting After being resolved on 10% SDS-PAGE gels, the protein samples were transferred onto nitrocellulose membrane in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) for 1 h at 100 V. The filters were then blocked in TTBS [20 mM Tris-HCl (pH 7.4), 500 mM NaCl and 0.3% Tween-20] containing 5% non-fat milk overnight at 4 °C and probed with primary antibodies diluted in TTBS containing 5% non-fat milk, monoclonal antibody against cyclin B1 (Upstate) diluted 1:1000, goat polyantibody against cyclin A2 (Santa Cruz) diluted 1:1000, monoclonal antibody against cyclin B2 (Santa Cruz) diluted 1:1000, rabbit serum against GFP (generated by injecting rabbit) diluted 1:5000.

Techniques: Control, Immunostaining, Expressing, Saline

Journal: Cell research

Article Title: Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis.

doi: 10.1038/cr.2008.11

Figure Lengend Snippet: Figure 4 The cyclin box of cyclin B1 is required for the localization to kinetochores. (A) Diagram showing several mutants of cyclin B1, each fused with the GFP tag at the N-terminus. (B) A truncated mutant of cyclin B1 lacking the cyclin box could not localize on kinetochores. The kinetochore localization of cyclin B1 is determined by its immunostaining patterns with paired dots on both sides of each chromosome. Truncated mutants, 1-90aa and 1-200aa of cyclin B1, expressed as GFP fusions could not localize on kinetochores in contrast to full-length cyclin B1. The accumulation of one truncation (150-433aa) on kinetochores was weaker than that of full-length cyclin B1. The kinetochore localization of cyclin B1 proteins is indicated with arrows. Bar = 5 µm. (C) Kinetochore localization of cyclin B1 mutants harboring deletions or point mutation at the N-terminus (green) on a chromosome spread. Cells were immunostained with anti-CenpA antibody to indicate kinetochores (red). Selected regions were magnified, showing the kinetochore localization of cyclin B1 mutants. Bar = 5 µm.

Article Snippet: Cell extracts and Western blotting After being resolved on 10% SDS-PAGE gels, the protein samples were transferred onto nitrocellulose membrane in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) for 1 h at 100 V. The filters were then blocked in TTBS [20 mM Tris-HCl (pH 7.4), 500 mM NaCl and 0.3% Tween-20] containing 5% non-fat milk overnight at 4 °C and probed with primary antibodies diluted in TTBS containing 5% non-fat milk, monoclonal antibody against cyclin B1 (Upstate) diluted 1:1000, goat polyantibody against cyclin A2 (Santa Cruz) diluted 1:1000, monoclonal antibody against cyclin B2 (Santa Cruz) diluted 1:1000, rabbit serum against GFP (generated by injecting rabbit) diluted 1:5000.

Techniques: Mutagenesis, Immunostaining

Journal: Cell research

Article Title: Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis.

doi: 10.1038/cr.2008.11

Figure Lengend Snippet: Figure 5 Cyclin B1 is required for the stable attachment of microtubules to kinetochores. (A) HeLa cells transfected with control or pSuper-cyclin B1 (cyclin B1 RNAi) were collected 48 h after transfection and analyzed by western blotting with cdc2 serving as the loading control (left). GFP-H2B and pSuper-cyclin B1 co-transfected (1:20) metaphase cells were immunostained with anti-cyclin B1 antibody (red); and the unaligned chromosomes are indicated with arrows (right). Bar = 10 µm. (B) The percent- age of metaphase cells with unaligned chromosomes was quantified in control (n > 500) or cyclin B1-depleted (n > 500) cells. Each experiment was repeated three times. Error bars represent SD. Arrow indicates unaligned chromosomes. Bar = 10 µm. (C) Images of cyclin B1-depleted HeLa cells stained for Bub1 (red). GFP-H2B was co-transfected with pSuper-cyclin B1 at a ratio of 1:20. Prometaphase and metaphase with unaligned chromosomes are shown. Selected regions were magnified to show the intensity of Bub1 on prometaphase chromosomes, unaligned chromosome and aligned chromosomes. Bar = 10 µm. (D) Control cells (control) and cells with depletion of cyclin B1 (pS-cyclin B1) were treated with ice-cold conditions for 10 min and fixed to be immunostained with anti-a-tubulin antibody (red) and observed by confocal microscopy. The merged image of z-stacks is shown in the upper panel and a sole deconvolution image of the same cell is shown in the lower panel. Bar = 10 µm. (E) Control and pS-cyclin B1 transfected cells were immunostained with anti-α-tubulin antibody (red) and were observed by confocal microscopy. GFP-Cenp A was transfected to show paired kinetochores. Selected regions were magnified and are shown. Bar = 10 µm. (F) Interkinetochore distance was determined by the distance of paired GFP-Cenp A signals. Over 100 pairs of kinetochores were scaled in at least 20 cells (average of three independent experiments). Error bars represent SD.

Article Snippet: Cell extracts and Western blotting After being resolved on 10% SDS-PAGE gels, the protein samples were transferred onto nitrocellulose membrane in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) for 1 h at 100 V. The filters were then blocked in TTBS [20 mM Tris-HCl (pH 7.4), 500 mM NaCl and 0.3% Tween-20] containing 5% non-fat milk overnight at 4 °C and probed with primary antibodies diluted in TTBS containing 5% non-fat milk, monoclonal antibody against cyclin B1 (Upstate) diluted 1:1000, goat polyantibody against cyclin A2 (Santa Cruz) diluted 1:1000, monoclonal antibody against cyclin B2 (Santa Cruz) diluted 1:1000, rabbit serum against GFP (generated by injecting rabbit) diluted 1:5000.

Techniques: Transfection, Control, Western Blot, Staining, Confocal Microscopy

Journal: Cell research

Article Title: Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis.

doi: 10.1038/cr.2008.11

Figure Lengend Snippet: Figure 6 Depletion of cyclin B1 delays chromosome alignment. (A) Still frames from time-lapse microscopy of control (control) or cyclin B1-depleted (pS-cyclin B1) HeLa/GFP-H2B cells are listed. Time is given in minutes. Arrow indicates unaligned chro- mosomes. Bar = 5 µm. (B) The duration of different periods from prophase to anaphase onset was calculated from time-lapse movies of 50 control and 78 cyclin B1-depleted cells. HeLa/GFP-H2B cells were monitored by time-lapse microscopy using a very low level of light intensity to determine the duration of NEBD to metaphase with all chromosomes aligned to the equator (NEBD to alignment). Error bars represent SD. (C) Histograms showing the percentages of mitotic cells that had progressed within the indicated time frames from prophase to complete chromosome alignment.

Article Snippet: Cell extracts and Western blotting After being resolved on 10% SDS-PAGE gels, the protein samples were transferred onto nitrocellulose membrane in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) for 1 h at 100 V. The filters were then blocked in TTBS [20 mM Tris-HCl (pH 7.4), 500 mM NaCl and 0.3% Tween-20] containing 5% non-fat milk overnight at 4 °C and probed with primary antibodies diluted in TTBS containing 5% non-fat milk, monoclonal antibody against cyclin B1 (Upstate) diluted 1:1000, goat polyantibody against cyclin A2 (Santa Cruz) diluted 1:1000, monoclonal antibody against cyclin B2 (Santa Cruz) diluted 1:1000, rabbit serum against GFP (generated by injecting rabbit) diluted 1:5000.

Techniques: Time-lapse Microscopy, Control

Journal: Cell research

Article Title: Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis.

doi: 10.1038/cr.2008.11

Figure Lengend Snippet: Figure 7 Depletion of cyclin B1 compromises the spindle checkpoint. (A) Still images of time-lapse microscopy of a HeLa/GFP- H2B cell transfected with pSuper-cyclin B1. Anaphase onset was between 78-81 min. Arrows indicate unaligned chromosomes. Time is given in minutes. Bar = 5 µm. (B) Still images of a representative HeLa/GFP-H2B cell transfected with pSuper-cyclin B1 treated with 200 nM nocodazole. Time is given in minutes. Bar = 5 µm. (C) A representative multinuclear cell caused by depletion of cyclin B1 was immunostained with anti-lamin B antibody to show the interphase nuclear membrane. Bar = 10 µm. (D) Diagram showing experimental protocols used in (E) and (F). (E) The effect of depletion of cyclin B1 on nocodazole-treated cells. Multinuclear cells were quantified in control (control, n > 500) or cyclin B1-depleted (pS-cyclin B1, n > 500) cells at each time point as indicated (average of three independent experiments). The time point at which cells were released from double thymidine block is indicated as 0 hour. The percentage of multinuclear cells at each time point listed in the figure is shown. (F) Phase contrast images of control and cyclin B1 depleted cells that were released from double thymidine block for 15 h. DMSO or MG132 was added at the 8 h time point with nocodazole (200 nM). Multinuclear cells are indicated with arrows. Bar = 100 µm.

Article Snippet: Cell extracts and Western blotting After being resolved on 10% SDS-PAGE gels, the protein samples were transferred onto nitrocellulose membrane in transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol) for 1 h at 100 V. The filters were then blocked in TTBS [20 mM Tris-HCl (pH 7.4), 500 mM NaCl and 0.3% Tween-20] containing 5% non-fat milk overnight at 4 °C and probed with primary antibodies diluted in TTBS containing 5% non-fat milk, monoclonal antibody against cyclin B1 (Upstate) diluted 1:1000, goat polyantibody against cyclin A2 (Santa Cruz) diluted 1:1000, monoclonal antibody against cyclin B2 (Santa Cruz) diluted 1:1000, rabbit serum against GFP (generated by injecting rabbit) diluted 1:5000.

Techniques: Time-lapse Microscopy, Transfection, Membrane, Control, Blocking Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Astaxanthin Prevents Tuberculosis-Associated Inflammatory Injury by Inhibiting the Caspase 4/11-Gasdermin-Pyroptosis Pathway

doi: 10.1155/2022/4778976

Figure Lengend Snippet: ASTA did not significantly change the period field of the MLE-12 cells induced by LPS. (a) Cell cycle after different treatments using flow cytometry. (b) Western blotting shows the protein expression of CCNA2, CCNB1, CCNE1, procaspase 3, and caspase 3 in MLE-12 cells. The data are presented as a mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01 compared with the control.

Article Snippet: Antibodies against cyclin A2 (CCNA2), cyclin B1 (CCNB1), and cyclin E1 (CCNE1) were purchased from HUABIO (Hangzhou, China).

Techniques: Flow Cytometry, Western Blot, Expressing, Control

Journal: Molecular Medicine Reports

Article Title: Downregulation of long non-coding RNA ANRIL promotes proliferation and migration in hypoxic human pulmonary artery smooth muscle cells

doi: 10.3892/mmr.2019.10887

Figure Lengend Snippet: ANRIL induces higher levels of proliferation in HPASMCs. (A) The protein level of PCNA was analyzed by western blotting after transfection with si-ANRIL; densitometric quantification of protein bands showed a significant increase in PCNA following 24 h of transfection with si-ANRIL (n=3). (B) 5-bromodeoxyuridine incorporation assay was performed to determine the proliferation of HPASMCs (n=5). (C) Immunofluorescence assay was used to detect the expression of Ki67. The red color denotes Ki67 staining by rhodamine, whereas the blue color denotes nucleus staining by DAPI (n=3). Scale bar=50 µm. *P<0.05, **P<0.01 and ***P<0.001. CON, normoxia control; HYP, hypoxia; hyp+si-NC, cells treated with negative control under hypoxic conditions; hyp+si-ANRIL, cells treated with si-ANRIL under hypoxic conditions; HPASMCS, human pulmonary artery smooth muscle cells; PCNA, proliferating cell nuclear antigen; ANRIL, antisense noncoding RNA in the INK4 locus; DAPI, 4,6-diamidino-2-phenylindole.

Article Snippet: Antibodies against proliferating cell nuclear antigen (PCNA), Cyclin A, Cyclin D and Cyclin E were purchased from Boster Biological Technology.

Techniques: Western Blot, Transfection, BrdU Incorporation Assay, Immunofluorescence, Expressing, Staining, Control, Negative Control

Journal: International journal of oncology

Article Title: Long non‑coding RNA CASC11 interacts with YBX1 to promote prostate cancer progression by suppressing the p53 pathway.

doi: 10.3892/ijo.2022.5400

Figure Lengend Snippet: Figure 7. Knockdown of YBX1 in prostate cancer cells reverses the carcinogenic effects of CASC11. YBX1 knockdown reversed the OE‑CASC11‑induced (A and B) proliferation (scale bar, 500 µm) and (C and D) migration (scale bar, 100 µm) of LNCaP and 22Rv1 cells. *P<0.05, **P<0.01. (E) Western blot analysis demonstrated that YBX1 knockdown abolished the OE‑CASC11‑induced promotion of Cyclin A2, CDK2 and CDK4 proteins. (F) Western blot analysis demon‑ strated that YBX1 overexpression reversed the sh‑CASC11‑induced increase in the expression levels of p21 and p53 proteins. *P<0.05, **P<0.01 vs. the control group; #P<0.05, ##P<0.01 vs. the OE‑CASC11 or sh‑CASC11 group. CASC11, cancer susceptibility candidate 11; NC, negative control; OE‑CASC11, CASC11 overexpression plasmid; sh, short hairpin; YBX1, Y‑box binding protein 1.

Article Snippet: The membranes were blocked with 5% nonfat dry milk at room temperature for 2 h and blotted with primary antibodies against Cyclin A2 (cat. no. 67955; 1:1,000; Cell Signaling Technology, Inc.), CDK2 (cat. no. 18048; 1:1,000; Cell Signaling Technology, Inc.), CDK4 (cat. no. 12790; 1:1,000; Cell Signaling Technology, Inc.), p53 (cat. no. 2527; 1:1,000; Cell Signaling Technology, Inc.), p21 (cat. no. 2947; 1:1,000; Cell Signaling Technology, Inc.), YBX1 (cat. no. ab76149; 1:1,000; Abcam) and GAPDH (cat. no. ab8245; 1:2,000; Abcam) at 4 ̊C overnight.

Techniques: Knockdown, Migration, Western Blot, Over Expression, Expressing, Control, Negative Control, Plasmid Preparation, Binding Assay