Journal: bioRxiv

Article Title: Comparative ASCL1 interactome analysis reveals CDK2-Cyclin A2 as suppressors of differentiation in MYCN-amplified neuroblastoma

doi: 10.1101/2025.11.11.687869

Figure Lengend Snippet: (A) Volcano plot of quantitative ASCL1 interactome analysis (qPLEX-RIME) between IMR ASCL1 and BE ASCL1 cell lines treated with 1 µg/mL dox for 24 hours, highlighting CDK and Cyclin proteins detected. In blue, are highlighted proteins associating with ASCL1 more in BE ASCL1 cells compared to IMR ASCL1 , and in red, are highlighted proteins associating with ASCL1 more in the IMR ASCL1 cells compared to BE ASCL1 . Proteins are considered enriched when |log2 fold change| >1 and adjusted p value < 0.05. (B) Dot plot comparing Total Proteome and ASCL1 qPLEX-RIME in BE ASCL1 and IMR ASCL1 cell lines treated with 1 µg/mL dox for 24 hours. In red are highlighted proteins enriched in binding to ASCL1 in IMR ASCL1 cells, and in blue the proteins enriched in binding to ASCL1 in BE ASCL1 cells (qPLEX-RIME). Labelled are all CDKs and Cyclins detected in the ASCL1 qPLEX-RIME. (C) Representative western blot analysis of ASCL1 protein in the chromatin fraction of BE ASCL1 cells. The cells were left untreated or treated with dox for 24 hours, in combination with small molecule CDK inhibitors of CDK4/6 (5 µM of Palbociclib, PB), CDK1 (16 µM of RO3360, RO) or CDK2 (8 µM of KO, KO). Λ Protein Phosphatase treatment was used as control for un-phosphorylated ASCL1. Two western blot lanes, between KO and λ Protein Phosphatase, were cut out the blot in PowerPoint. n=4. (D) Quantification of western blot analysis in (C), shown as percentage of phosphorylated and un-phosphorylated ASCL1. Mean ± SD, n=4. Unpaired t test, * p <= 0.05. See also .

Article Snippet: Cells were treated with CDK4/6 inhibitor Palbociclib (PB, Selleckchem) at 5 μM, CDK1 inhibitor RO3306 (RO, Selleckchem) at 16 μM and CDK2 inhibitor KO3861 (KO, Selleckchem) at 8 μM for 24 hours in combination with 1 μg/mL doxycycline.

Techniques: Binding Assay, Western Blot, Control

Journal: bioRxiv

Article Title: Comparative ASCL1 interactome analysis reveals CDK2-Cyclin A2 as suppressors of differentiation in MYCN-amplified neuroblastoma

doi: 10.1101/2025.11.11.687869

Figure Lengend Snippet: Phos-tag western blot analysis of in vitro kinase assay with translated HA-tagged human wild-type ASCL1 and human recombinant Cyclin-CDK complexes, such as CyclinB-CDK1, CyclinA-CDK2, CyclinE-CDK2 or CDK4-CyclinD1. n=3.

Article Snippet: Cells were treated with CDK4/6 inhibitor Palbociclib (PB, Selleckchem) at 5 μM, CDK1 inhibitor RO3306 (RO, Selleckchem) at 16 μM and CDK2 inhibitor KO3861 (KO, Selleckchem) at 8 μM for 24 hours in combination with 1 μg/mL doxycycline.

Techniques: Western Blot, In Vitro, Kinase Assay, Recombinant

Journal: Nucleic Acids Research

Article Title: Phosphorylation of Msx1 promotes cell proliferation through the Fgf9/18-MAPK signaling pathway during embryonic limb development

doi: 10.1093/nar/gkaa905

Figure Lengend Snippet: Phosphorylation of Msx1 at Ser136 is essential for its binding to and upregulation of Fgf9 and Fgf18 and further activation the MAPK signaling pathway, and CDK1 appears to be the kinase that performs this phosphorylation. ( A ) Western blotting to assess the effect of Msx1 phosphorylation sites on increases in the levels of p-Erk1/2, Fgf9 and Fgf18 by wild-type Msx1. ( B ) qRT-PCR assays to assess the effect of Msx1 phosphorylation sites on increases in the levels of Fgf9 and Fgf18 by wild-type Msx1. Values are the means ± SD. *** P < 0.0001. ( C ) ChIP-qPCR analysis to determine the impact of Ser136 mutants of Msx1 on Fgf9/18 binding. ChIP assays were performed with C2C12 cells overexpressing wild-type Msx1, Msx1 (S136A), Msx1 (S136D) or the control. ChIP-qPCR was then used to determine the relative enrichment of the Msx1 binding fragments of the Fgf9 and Fgf18 genes. The ChIP-qPCR data were analyzed to calculate the enrichment of the control or different Msx1 mutants relative to input respectively, and the control values were defined as 1 to calculate the fold enrichment of Msx1 or its mutants over the control. Values are the means ± SD. *** P < 0.0001, ** P < 0.001, * P < 0.01. ( D ) Diagram of the conserved residues of the CDK1 catalytic domain and the corresponding phosphorylated peptide sequence of Msx1. ( E ) Western blotting to examine the effect of Msx1 or its mutants and Ro3306 on the level of p-Erk1/2 in C2C12 cells. C2C12 cells overexpressing wild-type Msx1, Msx1(S136A), Msx1(S136D) or the control were treated with 10 μM Ro3306 or DMSO. Twenty hours later, the cells were harvested and lysed for western blotting, and the level of p-Erk1/2 was checked. ( F ) Western blotting to verify the specificity of Ser136 phosphorylated Msx1 (p-Msx1) antibody. Lysates from C2C12 cells overexpressing Flag-tagged Msx1 were incubated with calf intestinal alkaline phosphatase (CIAP) at 37°C for half an hour and subjected to immunoblot analysis with the indicated antibodies. ( G ) Western blotting to examine the effect of Ro3306 on the level of endogenous p-Msx1 in C2C12 cells. C2C12 cells were treated with 10 μM Ro3306 or DMSO. Twenty hours later, the cells were harvested and lysed for western blotting with the indicated antibodies.

Article Snippet: The CDK1 inhibitor RO-3306 was purchased from APExBio Technology.

Techniques: Phospho-proteomics, Binding Assay, Activation Assay, Western Blot, Quantitative RT-PCR, ChIP-qPCR, Control, Sequencing, Incubation

Journal: Nucleic Acids Research

Article Title: Phosphorylation of Msx1 promotes cell proliferation through the Fgf9/18-MAPK signaling pathway during embryonic limb development

doi: 10.1093/nar/gkaa905

Figure Lengend Snippet: Deficiency of Msx in the lateral plate mesoderm reduced bone formation. ( A ) Representative views of 4-week-old control and Msx1/2 MSC-specific knockout littermates. The black arrow indicates the forelimb. ( B ) μCT images of bones from 4-week-old wild-type and Msx1/2 MSC-specific knockout mice. ( C ) Alcian blue and ARS staining in the forelimbs from 4-week-old control and Msx1/2 MSC-specific knockout mice. (D, E) Alcian blue ( D ) and ALP ( E ) staining of limb sections from wild-type and Msx1/2 MSC-specific knockout mice at postnatal day 2 (P2), scale bar = 0.5 mm. ( F ) IF assays of limb sections from wild-type and Msx1/2 MSC-specific knockout mice at P2. Expression of Sox9 (green) and PCNA (red) was detected. DAPI was used to counterstain the nuclei. Scale bar = 0.2 mm. (G, H) Diagram summarizing the immunofluorescence data. The proportions of PCNA ( G )- and Sox9 ( H )-positive cells (n = 400) were calculated. Values are the means ± SD. *** P < 0.0001, ** P < 0.001. ( I ) CCK-8 assays to evaluate the proliferation of primary bone marrow MSCs from wild-type and Msx1/2 MSC-specific knockout mice. Values are the means ± SD. *** P < 0.0001, ** P < 0.001, * P < 0.01. ( J ) Western blotting to detect PCNA, p-Erk1/2, Fgf9 and Fgf18 levels in primary bone marrow MSCs from wild-type and Msx1/2 MSC-specific knockout mice. ( K ) Model of the promotion of cellular proliferation by Msx1. In the nucleus, Ser136 of Msx1 is phosphorylated by CDK1. Phosphorylated Msx1 binds to Fgf9 and Fgf18 to activate their transcription. Upregulated Fgf9 and Fgf18 are shuttled to the extracellular matrix, where they bind to Fgfrs to activate the MAPK signaling pathway and promote the phosphorylation of Erk1/2. p-Erk1/2 then promotes cell proliferation in a variety of ways.

Article Snippet: The CDK1 inhibitor RO-3306 was purchased from APExBio Technology.

Techniques: Control, Knock-Out, Staining, Expressing, Immunofluorescence, CCK-8 Assay, Western Blot, Phospho-proteomics

Journal: Cellular signalling

Article Title: Cyclin-Dependent Kinase 1-Mediated Phosphorylation of YES Links Mitotic Arrest and Apoptosis during Antitubulin Chemotherapy

doi: 10.1016/j.cellsig.2018.09.007

Figure Lengend Snippet: A, HeLa and U2OS cells were treated with taxol. RO3306 (CDK1 inhibitor) or Purvalanol A (CDK1/2/5 inhibitor) was added (with MG132 to block mitotic exit by stabilizing cyclin B) into the cells 1.5 h before harvesting the cells. Total cell lysates were subjected to Western blotting with the indicated antibodies. B, A diagram shows the N-terminal sequence of YES with 5 S/TP consensus. C, In vitro kinase assays with purified CDK1/cyclin B complex (New England Biolab). D, In vitro kinase assays were done as in C except anti-phospho-YES antibodies were used.

Article Snippet: The MK5108 (Aurora-A inhibitor) was from Merck (NJ, USA), and RO3306 (CDK1 inhibitor) and Purvalanol A (CDK1/2/5 inhibitor) were from ENZO Life Sciences (NY, USA).

Techniques: Blocking Assay, Western Blot, Sequencing, In Vitro, Purification

Journal: Cellular signalling

Article Title: Cyclin-Dependent Kinase 1-Mediated Phosphorylation of YES Links Mitotic Arrest and Apoptosis during Antitubulin Chemotherapy

doi: 10.1016/j.cellsig.2018.09.007

Figure Lengend Snippet: A, HEK293T cells were transfected with Flag-tagged YES and non-phosphorylatable mutant (YES-5A). Cells were treated with taxol at 48 h post-transfection. Total cell lysates were probed with the indicated antibodies. Increased p-Aurora-A,-B mark cells in mitosis. B, HEK293T cells were transfected with Flag-YES. At 30 h post-transfection, the cells were treated with taxol for 16 h. RO3306 (5 µM) or Purvalanol A (10 µM) was added to cells 1.5 h before harvesting as indicated. Proteasome inhibitor MG132 was also added (together with inhibitors) to prevent cyclin B from degradation and cells from exiting from mitosis. Total cell lysates were probed with anti-phospho-YES and subsequent anti-Flag antibodies. C, YES knockout (KO) HeLa cells were stably transduced with YES-WT or YES-5A. Total cell lysates from these cell lines were treated with taxol and probed with the indicated antibodies. S.E.: short exposure; LE: long exposure. D, HEK293T cells were transfected with Flag-tagged YES and non-phosphorylatable mutant (YES-5A). Cells were treated with taxol at 48 h post-transfection and YES proteins were immunoprecipitated (with Flag) antibody and analyzed with the indicated antibodies.

Article Snippet: The MK5108 (Aurora-A inhibitor) was from Merck (NJ, USA), and RO3306 (CDK1 inhibitor) and Purvalanol A (CDK1/2/5 inhibitor) were from ENZO Life Sciences (NY, USA).

Techniques: Transfection, Mutagenesis, Knock-Out, Stable Transfection, Transduction, Immunoprecipitation

Journal: Journal of Virology

Article Title: Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication

doi: 10.1128/JVI.00402-19

Figure Lengend Snippet: ATR kinase and CDKs promote SMARCAL1 degradation following Ad5 and Ad12 infection. A549 cells were either mock infected or infected with 10 PFU/cell of wt Ad5 (A and C) or wt Ad12 (B and D). Cells were then incubated in the absence or presence of ATR inhibitor (AZD6738 [ATRi], 1 μM; A and B) or ATR and CDK inhibitors (AZD6738, 1 μM and RO-3306 [CDKi], 9 μM; C and D) and harvested at the appropriate times postinfection. Cell lysates were then separated by SDS-PAGE and subjected to WB for SMARCAL1, p53, E1B-55K, and β-actin. h.p.i, hours postinfection. Data are representative of three independent experiments.

Article Snippet: The ATR inhibitor AZD6738 and the CRL inhibitor MLN4924 were purchased from Cayman chemicals, while the CDK inhibitor RO-3306 was purchased from Merck Millipore.

Techniques: Infection, Incubation, SDS Page