Journal: Frontiers in Oncology

Article Title: PTEN loss promotes Warburg effect and prostate cancer cell growth by inducing FBP1 degradation

doi: 10.3389/fonc.2022.911466

Figure Lengend Snippet: CDK inhibitor blocks FBP1 degradation induced by PTEN loss (A) WB was carried out using the whole-cell lysate and co-IP samples obtained from 293T cells transfected with the indicated constructs. β-Tubulin was used as a WB loading control. (B, C) Western blot (B) and quantitative RT-PCR (C) analysis in 22Rv1 and DU145 cells infected with lentivirus expressing control or PTEN-specific shRNAs for 48 hours and treated with Roscovitine for 24 hours. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (D, E) WB (D) and quantitative RT-PCR (E) were carried out in MEFs generated from Pten p/p conditional mice infected with or without lentivirus expression CMV-driven Cre and treated with Roscovitine for 24 hours. All quantitative data are shown as Mean ± SD (n=3). Student’s t test. n.s., not significant. (F, G) WB and quantification of WB bands were performed in 22Rv1 (F) and DU145 cells (G) treated with Roscovitine for 48 hours followed by treatment with 50 μg/ml cycloheximide (CHX) for different periods of time. At each time point, the intensity of FBP1 was normalized to the intensity of β-Tubulin (a WB loading control) first and then to the value at the 0-h time point.

Article Snippet: The next day, the cells were treated with vehicle or Roscovitine (10 μM, Cat# HY-30237, MedChemExpress).

Techniques: Co-Immunoprecipitation Assay, Transfection, Construct, Control, Western Blot, Quantitative RT-PCR, Infection, Expressing, Generated

Journal: Virulence

Article Title: Cyclin-dependent kinase 5 negatively regulates antiviral immune response by disrupting myeloid differentiation primary response protein 88 self-association.

doi: 10.1080/21505594.2023.2223394

Figure Lengend Snippet: Figure 3. The regulation of CDK5 in IFN-β production is independent of its kinase activity. Note: A. Flag-CDK5 was co-transfected with P35-V5 into 293T cells. After infection with VSV-GFP for 2, 4 or 6 hours, the cells were lysed and anti-Flag beads were used for immunoprecipitation. B. After 8 hours of VSV-GFP (MOI = 0.1) infection, the mRNA expression of IFN-β in A549 cells overexpressing Flag-CDK5, Flag-CDK5 kinase inactive mutant (Flag-mutant) or EV was analysed by qRT-PCR assay C. Left panel: Flag-CDK5, Flag-mutant or EV plasmids were transfected into A549 cells, followed by 8-hour infection with VSV-GFP (MOI = 0.1). Western blotting was performed to determine the phosphorylated or total protein of IRF3. Right panel: Quantitative analysis of the relative abundances of P-IRF3 normalized to that of total IRF3. D-E. A549 cells were pre-treated with CDK5 kinase inhibitor Roscovitine (ROSC) for 30 minutes, followed by VSV-GFP (MOI = 0.1) infection for 8 hours with ROSC. Western blotting was performed to determine the phosphorylated or total protein of IRF3 (D), and IFN-β expression was analysed by qRT-PCR assay (E). All graph values are presented as means ± SE, n = 3. *P < 0.05, **P < 0.01.

Article Snippet: The reagents used were as follows: 3-Methyladenine (3 MA; 189490, Sigma), MG132 (474790, Millipore), Roscovitine (SC−24002, Santa Cruz), zymosan (Z4250, Sigma), lipopolysaccharide (LPS; L2880, Sigma), imiquimod (IMQ; HY-B0180A, MCE).

Techniques: Activity Assay, Transfection, Infection, Immunoprecipitation, Expressing, Mutagenesis, Quantitative RT-PCR, Western Blot

Journal: PLOS ONE

Article Title: Palmitoyl-L-carnitine induces tau phosphorylation and mitochondrial dysfunction in neuronal cells

doi: 10.1371/journal.pone.0313507

Figure Lengend Snippet: (A) shows changes in protein levels of pGSK-3β (S9), GSK-3β, CDK5, and p25 after treatment with palmitoyl-L-carnitine in SH-SY5Y cells. pGSK-3β (S9) indicates GSK-3β phosphorylated at serine 9. Full blots are provided in . (B) Histogram illustrating changes in protein levels of pGSK-3β (S9), CDK5, and p25 after treatment with palmitoyl-L-carnitine in SH-SY5Y cells, shown as mean ± standard error of the mean (SEM; n = 3). GSK-3β phosphorylation levels were normalized to the total GSK-3β protein. (C) shows changes in protein levels of pTau (T181), pTau (S262), PHF-1, total tau, GSK-3β, CDK5, and p25 after treatment with tau kinase inhibitors in palmitoyl-L-carnitine-treated SH-SY5Y cells. pTau (T181), pTau (S262), and PHF-1 represent tau phosphorylated at threonine 181, serine 262, and serine 396/404, respectively. SB216763, Roscovitine, and PD150606 are GSK-3β inhibitor, CDK5 inhibitor, and calpain inhibitor, respectively. Full blots are provided in . (D) Histogram illustrating changes in protein levels of pTau (T181), pTau (S262), PHF-1, total tau, GSK-3β, CDK5, and p25 after treatment with tau kinase inhibitors in palmitoyl-L-carnitine-treated SH-SY5Y cells, shown as mean ± standard error of the mean (SEM; n = 3). Tau phosphorylation levels were normalized to the total tau protein. In (A) and (D) , BSA and BSA-PC represent bovine serum albumin and BSA-conjugated palmitoyl-L-carnitine, respectively. Statistical significance was determined using an unpaired two-tailed t-test with Welch’s correction and an ordinary two-way ANOVA with Tukey’s multiple comparison test; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: To assess the effects of kinase inhibitors, cells were treated simultaneously with 5 μM GSK-3β inhibitor SB216763 (Tocris), 5 μM CDK5 inhibitor Roscovitine (Tocris), or 5 μM calpain inhibitor PD150606 (Tocris), each combined with 5 μM BSA-PC for 24h.

Techniques: Phospho-proteomics, Two Tailed Test, Comparison

Journal: PLOS ONE

Article Title: Palmitoyl-L-carnitine induces tau phosphorylation and mitochondrial dysfunction in neuronal cells

doi: 10.1371/journal.pone.0313507

Figure Lengend Snippet: A schematic illustration showing the mechanism by which palmitoyl-L-carnitine induces tau phosphorylation in SH-SY5Y neurons. Palmitoyl-L-carnitine causes calcium overload by closely interacting with mitochondrial malfunction, including the fission process. This increased calcium overload activates tau kinases (GSK-3β and CDK5/p25), leading to significant tau phosphorylation. Therefore, elevated serum levels of palmitoyl-L-carnitine are likely to contribute significantly to the development of AD pathology with aging.

Article Snippet: To assess the effects of kinase inhibitors, cells were treated simultaneously with 5 μM GSK-3β inhibitor SB216763 (Tocris), 5 μM CDK5 inhibitor Roscovitine (Tocris), or 5 μM calpain inhibitor PD150606 (Tocris), each combined with 5 μM BSA-PC for 24h.

Techniques: Phospho-proteomics

Journal: bioRxiv

Article Title: Extracellular vesicles adhere to cells predominantly through the interaction of CD151-associated integrin heterodimers and GM1 with laminin

doi: 10.1101/2024.04.11.589011

Figure Lengend Snippet: Talin-1 in sEVs does not regulate the binding affinity of integrins for laminin. (A) Western blot analysis of PC3 cell-derived sEVs after talin-1 KD by siRNA or overexpression of talin-1. (B, C, and D) The fluorescence images (B) and the numbers of PC3-sEVs attached to glass coated with laminin before and after (C) talin-1 KD or (D) overexpression of talin-1. Halo7-integrin β1 in sEVs was labeled with SF650T. (E, F, and G) The fluorescence images (E) and the numbers of CD63-labeled sEVs attached to glass coated with laminin before and after (F) talin-1 KD and (G) overexpression of talin-1. (H) Western blot analysis of the phosphorylation of Ser425 on talin-1 in PC3 cells and sEVs. Roscovitine: an inhibitor of CDK5 that phosphorylates Ser425 of talin-1.

Article Snippet: For roscovitine treatment, cells were incubated with 20 μM roscovitine (LKT Laboratories) in FBS-free medium.

Techniques: Binding Assay, Western Blot, Derivative Assay, Over Expression, Fluorescence, Labeling, Phospho-proteomics

Journal: Molecules

Article Title: Several Human Cyclin-Dependent Kinase Inhibitors, Structurally Related to Roscovitine, As New Anti-Malarial Agents

doi: 10.3390/molecules190915237

Figure Lengend Snippet: IC 50 values for Pf 3D7 and Pf 7G8 for the six mammalian kinase inhibitors and the three control molecules. These values were estimated by non-linear regression analysis using an inhibitory sigmoid E max model. All assays were run in triplicates.

Article Snippet: Basically, ( R )-roscovitine, a 2,6,9-trisubstituted purine, is a very promising molecule developed by Cyclacel Pharmaceuticals which reached phases 2 and 2b clinical trials against different types of cancers, and phase 1 clinical trial against glomerulonephritis.

Techniques: Control