Journal: Acta Pharmacologica Sinica

Article Title: LW-213, a newly synthesized flavonoid, induces G2/M phase arrest and apoptosis in chronic myeloid leukemia

doi: 10.1038/s41401-019-0270-4

Figure Lengend Snippet: LW-213 induced G2/M phase arrest in CML cell lines. a K562 and K562r cells were treated with the indicated concentrations of LW-213 for 12 and 24 h. Cell cycle arrest was determined by PI staining. b , c After treatment with LW-213, the proportions of cells in each cell cycle phase were summarized. Data represent the mean ± S.E.M. from three independent experiments. Asterisks denote statistically significant differences compared with untreated cells (* P < 0.05, ** P < 0.01). d , e After treatment with 0 and 15 μM LW-213 for 24 h, K562 and K562r cells were stained with β-tubulin antibody (labeled with Alexa Fluor 488 goat anti-rabbit lgG antibody; green fluorescence) and PI (red fluorescence). The number of cells in M phase was quantified in three experiments, with 200 cells per condition. Asterisks denote statistically significant differences compared with untreated cells (* P < 0.05). f , g Levels of Cyclin B1, CDC2 and p-CDC2 in K562 and K562r cells were analyzed by Western blots after treatment with LW-213 for 12 and 24 h, respectively. β-Actin was used as a loading control. The results are representative of three independent experiments. Data represent the mean ± S.E.M. from three independent experiments. Asterisks denote statistically significant differences compared with untreated cells (* P < 0.05, ** P < 0.01)

Article Snippet: Primary antibodies for Cyclin B1, CDC2, p-CDC2 (Y15), β-tubulin, MCL-1, p-CDK9 (Thr186), AKT, p-AKT (Ser473), β-actin, BCL-2, STAT5, STAT3, p-STAT5 (Y694), p-RNAPII-S2, p-RNAPII-S5, ABL1, LC3, P62/SQSTM1, caspase 3, and caspase 9 were obtained from ABclonal Technology (Wuhan, China).

Techniques: Staining, Labeling, Fluorescence, Western Blot, Control

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Schematic illustration of bis-DbTACs design, which is based on DbTACs. b Schematic illustration of three ligand covalent sites of bis-DbTACs equivalent to a DNA tetrahedral scaffold with three polyA domains. Au NPs (5, 10, and 15 nm) correspond to CRBN, CDK9, and CDK6 ligands, respectively. c Cartoon and representative TEM images of bis-DbTACs equivalents. Scale bars are 75 Å and 200 Å, respectively. d WB analysis of the selectively targeted degradation ability of bis-DbTACs at different concentrations and semiquantitative analysis of the grayscale. The error bars indicate the mean ± SD values; n = 3. e Immunofluorescence double-staining images of HepG2 cells treated with/without bis-DbTACs were recorded by confocal laser scanning microscopy. The cell nucleus was stained with DAPI. CDK6 and CDK9 proteins were labeled with anti-CDK6 and anti-CDK9 antibodies, respectively. Scale bars, 10 μm.

Article Snippet: The primary antibody used was mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:100), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:100).

Techniques: Immunofluorescence, Double Staining, Confocal Laser Scanning Microscopy, Staining, Labeling

Journal: American Journal of Cancer Research

Article Title: Cdc14B/Cyclin B1 signaling modulates the pathogenesis of sonic hedgehog subtype medulloblastoma

doi: 10.62347/CVAY8707

Figure Lengend Snippet: Shh induces nuclear translocation of Cyclin B1 via atypical Hedgehong signaling. A, B. Daoy cells were treated with or without Shh (100 ng/ml) for 24 h, and were subjected to immunofluorescence staining. Scale bar = 10 μm. C, D. Daoy cells were treated with or without Shh (100 ng/ml) for 24 h, and the extracted cytoplasmic (CE) and nuclear (NE) proteins were prepared for Western blot analyses. E, F. Daoy cells were treated with or without Shh (100 ng/ml) for 24 h, and the cell lysates were prepared and used for immunoprecipitation assays. G, H. Daoy cells were transfected with Cyclin B1 siRNAs and subjected to Western blot and qPCR validation for gene silencing. I. siCyclin B1 Daoy cells and control group were both treated with Shh and were performed to CCK8-assays. Data are representative images of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Sections were then blocked in 5% BSA for 30 minutes and incubated with primary antibodies against Cyclin B1 (PA5-32372, Abcam), (CST), Cdc14B (ab203675, Abcam), p-Cdc25C (Thr48) (bs-3482R, Thermo Fisher) or p-CDK1 (Tyr15) (PA5-121257, Thermo Fisher) at 4°C overnight.

Techniques: Translocation Assay, Immunofluorescence, Staining, Western Blot, Immunoprecipitation, Transfection, Control

Journal: American Journal of Cancer Research

Article Title: Cdc14B/Cyclin B1 signaling modulates the pathogenesis of sonic hedgehog subtype medulloblastoma

doi: 10.62347/CVAY8707

Figure Lengend Snippet: Cyclin B1 was stabilized due to Cdc14B deregulation in MB cells. A, B. MB tissues were isolated from Neurod2-SmoA transgenic mice (Jackson Laboratory, Stock #: 008831), and mouse CGNPs were obtained from neonatal mice. The homogenates of MB tissues and the lysates of CGNPs were prepared for Western blot and qPCR analyses. C, D. Daoy cells were transfected with the expressing constructs for GFP, wild-type or the enzyme activity-deficient mutant of Cdc14B, and were subjected to Western blot and qPCR analyses. E. Western blot analyses of p-Cdc25C, p-CDK1 and Cyclin B1 in Daoy cells with different overexpression. F. qPCR analyses of Cdc14B and Cyclin B1 in Daoy cells with different overexpression. G, H. GFP-, Cdc14B- and mutCdc14B- overexpressing Daoy cells were treated with 50 μg/ml CHX for indicated times, and were subjected to Western blot analyses. Data are representative images or are expressed as the means ± SD of three independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Article Snippet: Sections were then blocked in 5% BSA for 30 minutes and incubated with primary antibodies against Cyclin B1 (PA5-32372, Abcam), (CST), Cdc14B (ab203675, Abcam), p-Cdc25C (Thr48) (bs-3482R, Thermo Fisher) or p-CDK1 (Tyr15) (PA5-121257, Thermo Fisher) at 4°C overnight.

Techniques: Isolation, Transgenic Assay, Western Blot, Transfection, Expressing, Construct, Activity Assay, Mutagenesis, Over Expression

Journal: American Journal of Cancer Research

Article Title: Cdc14B/Cyclin B1 signaling modulates the pathogenesis of sonic hedgehog subtype medulloblastoma

doi: 10.62347/CVAY8707

Figure Lengend Snippet: Cdc14B/Cyclin B1 signaling controls MB cell proliferation. (A, B) Cdc14B-overexpression Daoy cells were transfected with expressing constructs for Cyclin B1 and subjected to Western blot and qPCR validation. (C) Different overexpression groups of Daoy cells were treated with Shh (100 ng/ml), with wildtype Daoy cells as control, were subjected to CCK-8 assays for proliferation ability. (D-G) Different overexpression groups of Daoy cells were treated with Shh (100 ng/ml), with wildtype Daoy cells as control, and subjected to immunofluorescence staining (D, E) and plate colony formation assays (F, G). Data are representative images or are expressed as the means ± SD of three independent experiments. Scale bar = 10 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Sections were then blocked in 5% BSA for 30 minutes and incubated with primary antibodies against Cyclin B1 (PA5-32372, Abcam), (CST), Cdc14B (ab203675, Abcam), p-Cdc25C (Thr48) (bs-3482R, Thermo Fisher) or p-CDK1 (Tyr15) (PA5-121257, Thermo Fisher) at 4°C overnight.

Techniques: Over Expression, Transfection, Expressing, Construct, Western Blot, Control, CCK-8 Assay, Immunofluorescence, Staining

Journal: American Journal of Cancer Research

Article Title: Cdc14B/Cyclin B1 signaling modulates the pathogenesis of sonic hedgehog subtype medulloblastoma

doi: 10.62347/CVAY8707

Figure Lengend Snippet: Cdc14B/Cyclin B1 signaling regulates in vivo development of Shh subtype MB. (A, B) MB tissues from Neurod2-SmoA transgenic mice were sliced and used for immunohistochemical staining. Scale bar = 10 μm. (C, D) Immunohistochemical staining was performed using microarrays of human MB specimens (C). The staining results were scored and subjected to Pearson correlation analysis (D). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Sections were then blocked in 5% BSA for 30 minutes and incubated with primary antibodies against Cyclin B1 (PA5-32372, Abcam), (CST), Cdc14B (ab203675, Abcam), p-Cdc25C (Thr48) (bs-3482R, Thermo Fisher) or p-CDK1 (Tyr15) (PA5-121257, Thermo Fisher) at 4°C overnight.

Techniques: In Vivo, Transgenic Assay, Immunohistochemical staining, Staining

Journal: American Journal of Cancer Research

Article Title: Cdc14B/Cyclin B1 signaling modulates the pathogenesis of sonic hedgehog subtype medulloblastoma

doi: 10.62347/CVAY8707

Figure Lengend Snippet: Schematic representation for the regulatory role of Cdc14B/Cyclin B1 in Shh-driven development of MB. Shh engagement on the cognate receptor Patched 1 causes the dissociation of Cyclin B1 from the receptor, allowing its nuclear translocation and transactivation of cell division-related genes. Meanwhile, Cdc14B is deficient in MB cells, which leads to the hyperactivation of its dephosphorylating substrate Cdc25C, reduced phosphorylation of CDK1 and stabilized complex of Cyclin B1/CDK1. The complex thus contributes to excessive proliferation through propelling cell cycle progression.

Article Snippet: Sections were then blocked in 5% BSA for 30 minutes and incubated with primary antibodies against Cyclin B1 (PA5-32372, Abcam), (CST), Cdc14B (ab203675, Abcam), p-Cdc25C (Thr48) (bs-3482R, Thermo Fisher) or p-CDK1 (Tyr15) (PA5-121257, Thermo Fisher) at 4°C overnight.

Techniques: Translocation Assay