cdk5 inhibitor roscovitine  (Tocris)

Journal: Bioactive Materials

Article Title: Mechanically sensitized hydrogel microspheres trigger membrane receptor switch for cartilage repair

doi: 10.1016/j.bioactmat.2026.03.017

Figure Lengend Snippet: Mechanism of integrin receptor activation induced by membrane receptor switch. A) Schematic representation of Calpain protein activity verified using fluorophores. B) The effectiveness of the fluorescence method was verified based on different groups. C) Calpain protein activity in MSCs subjected to different treatments. D) The expression of CDK5 and p-Talin head detected by Western blot analysis. E) Quantitative analysis of the Western blot results for the CDK5 and p-Talin head protein. F) The expression of p-Talin head and p-Talin detected by Western blot analysis. G) Quantitative analysis of the Western blot results for the p-Talin head protein. H) Quantitative analysis of the Western blot results for the p-Talin protein. I) Schematic diagram of Talin head and integrin β interaction verified by SPR technique. J) The association-dissociation curves of Talin head and integrin β1. K) Integrin β1 activation was verified by flow cytometry. L) Quantitative analysis of fluorescence intensity by flow cytometry. M) The expression of FAK and p-FAK detected by Western blot analysis. N) The quantification of FAK phosphorylation levels. O) Mechanism of integrin receptor activation induced by membrane receptor switch. (∗ symbol represents comparison with HAMA group, # symbol represents comparison with OBNC group). (ns: non-significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ## P < 0.01, ### P < 0.001).

Article Snippet: TRPC1 inhibitor (0.3 nM, Pico145, CAS No. 1628287-16-0), TRPM7 inhibitor (1.0 μM, VPC4, CAS No. 945604-76-2), TRPV2 inhibitor (5.0 μM, compound IV2-1, CAS No. 2242724-49-6), TRPM4 inhibitor (1.5 μM, CBA, CAS No. 351424-20-9), PIEZO1 inhibitor (2.5 μM, GsMTx4, CAS No. 1209500-46-8), integrin αvβ5 inhibitor (8.0 nM, Compound 12, CAS No.: 2615912-33-7), integrin αvβ1 inhibitor (0.3 nM, Compound C8, CAS No. 1689540-62-2), integrin α5β1 inhibitor (10 μM, ATN-161, 904763-27-5), and CDK5 inhibitor (5 nM, CDK5-IN-1, 2,639,540-19-3) were purchased from MCE Biotechnology Co., LTD. After the MSCs were treated, the cRGD solution was added at a concentration of 1:200 and incubated in the dark for 15 min, and the results were observed by fluorescence microscopy.

Techniques: Activation Assay, Membrane, Activity Assay, Fluorescence, Expressing, Western Blot, Flow Cytometry, Phospho-proteomics, Comparison

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: Expression levels of E2F1 and CDK5 in microglia after CIRI. (A, B) RT‐qPCR experiments assessing the expression levels of E2F1 and CDK5 in the brain tissue of CIRI mice. (C–F) Immunofluorescence experiments detected the expression levels of E2F1 (C, D) and CDK5 (E, F) in the brain tissues of mice 24 h after CIRI. (G, H) RT‐qPCR experiments measuring the expression levels of E2F1 and CDK5 in microglia after OGD/R, scale bars = 25 µm. (I, L) Immunofluorescence experiments investigating the expression levels of E2F1 (I, J) and CDK5 (K, L) in microglia after OGD/R. Scale bars = 25 µm. Each group consisted of six mice, and all cellular experiments were repeated three times. * p < .05, p ** < .01, *** p < 0.001.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: Transcriptional regulation of CDK5 by E2F1 in microglia. (A) Schematic diagram of the dual‐luciferase reporter gene experiment (Created with BioRender.com). (B) RT‐qPCR detection of E2F1 expression levels in cells after overexpression or silencing of E2F1. (C) RT‐qPCR detection of CDK5 expression levels in cells after overexpression or silencing of E2F1. (D) Dual‐luciferase assay to detect the effect of E2F1 on CDK5 promoter transcriptional activity: based on lentivirus‐mediated silencing and overexpression of E2F1 in 293T cells (oe‐NC, oe‐E2F1, sh‐NC, sh‐E2F1), co‐transfected with the dual‐luciferase reporter gene vector containing the CDK5 promoter sequence and its binding site mutant, luciferase activity was measured 48 h posttransfection and normalized to Renilla. (E) ChIP experiment to detect the enrichment of E2F1 on the CDK5 promoter. (F, G) TEM image showing mitochondrial morphology in oligodendrocytes. Scale bar = 500 nm. (H, I) Fluorescence images and related quantitative data showing Mito‐ROS MitoSOX fluorescence in oligodendrocytes of each group. Scale bar = 25 µm. (J, K) Immunofluorescence detection of mitochondrial morphology in oligodendrocytes of each group, red marks mitochondria, blue marks nuclei. Scale bar = 15 µm. (L, M) DCFH‐DA staining to detect ROS production in oligodendrocytes of each group. Scale bar = 25 µm. All cellular experiments were conducted thrice, * p < .05, ** p < .01.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques: Luciferase, Quantitative RT-PCR, Expressing, Over Expression, Activity Assay, Transfection, Plasmid Preparation, Sequencing, Binding Assay, Mutagenesis, Fluorescence, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: CDK5 regulation of DRP1 phosphorylation affects microglia mitochondrial fission and ROS accumulation. (A) TEM image displaying mitochondria in the cerebral cortex post‐CIRI along with quantitative data on mitochondrial length and cristae density per group. Scale bars = 2 µm/500 nm. (B) DCFH‐DA staining assessing ROS production in the cerebral cortex post‐CIRI. Scale bar = 25 µm. (C) Western blot determining the activation status of DRP1, with β‐actin, COX IV, and Tubulin serving as internal references for total, mitochondrial, and cytosolic fractions, respectively. (D, E) Immunofluorescence detecting the co‐localization of DRP1 and mitochondrial marker protein COX IV. Scale bar = 15 µm; (F, G) TEM image featuring mitochondrial morphology in microglia. Scale bar = 500 nm. (H, I) Fluorescence images and corresponding quantitative data showing MitoSOX fluorescence in microglia of each group. Scale bar = 25 µm. Each group comprised six mice, and all cellular experiments were repeated three times. * p < 0.05.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques: Staining, Western Blot, Activation Assay, Immunofluorescence, Marker, Fluorescence

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: Toxic effects of E2F1/CDK5 regulating DRP1 on neurons. (A) RT‐qPCR analysis of E2F1 and CDK5 expression levels in cells. (B, C) Western blot examining the activation status of DRP1, with β‐actin, COX IV, and Tubulin used as internal references for total, mitochondrial, and cytosolic fractions. (D) Morphological changes in neurons in each group. Scale bar = 25 µm. (E) CCK‐8 assay measuring the proliferative activity of neurons in each group. (F, G) Tunel staining assessing apoptosis in neurons of each group. Scale bar = 50 µm. (H) Quantification of LDH release indicating neuronal cell death. All cellular experiments were conducted thrice. * p < .05.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Activation Assay, CCK-8 Assay, Activity Assay, TUNEL Assay, Staining

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: Impact of E2F1 silencing in microglia on neurobehavioral functions in mice following CIRI injury. (A) RT‐qPCR analysis of E2F1 and CDK5 expression levels in brain tissue. (B, C) Western Blot analysis of DRP1 activation in brain tissue. (D) Quantitative assessment of TCC staining and infarct area in the mouse brain. (E) H&E staining of the cerebral cortex in mice. Scale bar = 50/25 µm. (F) Evaluation of neurological deficits in mice through the mNSS analysis. (G) Delay in reaching the hidden platform on the 6th day for mice. (H) Time spent in the target quadrant during the probe trial on the 7th day in seconds. (I) Number of crossings over the target platform location by mice on the 7th day during the probe trial. (J) Representative swim paths of mice on the 7th day. (K) Percentage of time spent exploring the novel object in the novel object recognition test phase. Each group comprises six mice, * p < .05.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Activation Assay, Staining

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: Schematic representation of the mechanism by E2F1/CDK5/DRP1 mediating microglial mitochondrial division and autophagy impact on CIRI.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques:

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: Translational neurodegeneration

Article Title: Neuronal double-stranded DNA accumulation induced by DNase II deficiency drives tau phosphorylation and neurodegeneration.

doi: 10.1186/s40035-024-00427-8

Figure Lengend Snippet: Fig. 2 DNase II deficiency promotes tau phosphorylation in primary hippocampal neurons through activation of the CDK5, CaMKII and PP2A signaling pathway. a Representative images of AT8 (red) and pThr231 (red) fluorescence staining in primary hippocampal neurons infected with shDNase2a or shCON. Scale bars, 10 μm. b Quantification of AT8 and pThr231 fluorescent areas in (a) by Image J software. c Western blotting of AT8 and pThr231 in the primary hippocampal neurons infected with shDNase2a or shCON. d Quantitation of the levels of AT8 and pThr231 in (c). e Western blotting of CDK5, Calpain2, p25, p35, CaMKII-α, p-CaMKII-α, PP2A, p-PP2A, CIP2A, GSK-3β, p-GSK-3β, Chk1, p-Chk1, Chk2 and p-Chk2 in the primary hippocampal neurons infected with shDNase2a or shCON. f Quantitation of the bands in (e) by the Image J software. g Representative images of pSer416 (cyan) fluorescence staining in primary hippocampal neurons infected with shDNase2a or shCON. Scale bars, 10 μm. h Quantification of pSer416 fluorescent area in (g) by the Image J software. i Western blotting of pSer416 in the primary hippocampal neurons infected with shDNase2a or shCON. j Quantitation of the levels of pSer416 in (i). In a, c, e, g and i, data are representative of three independent experiments. In b, d, f, h, and j, data were pooled from three independent experiments. Mean ± SEM, unpaired t-test with two-tailed P values was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant

Article Snippet: Meanwhile, at 48 h after infection, the cells were treated with 2.5 μM roscovitine [28] (MedChemExpress, #HY-30237) for 24 h to inhibit CDK5, or 5 μM DT061 [29] (MedChemExpress, #HY-112929) for 24 h to activate PP2A.

Techniques: Phospho-proteomics, Activation Assay, Fluorescence, Staining, Infection, Software, Western Blot, Quantitation Assay, Two Tailed Test

Journal: Translational neurodegeneration

Article Title: Neuronal double-stranded DNA accumulation induced by DNase II deficiency drives tau phosphorylation and neurodegeneration.

doi: 10.1186/s40035-024-00427-8

Figure Lengend Snippet: Fig. 6 Neuronal DNase II overexpression prevents cognitive deficits and tau phosphorylation in Tau-P301S mice. a, b The time spent and entries in the new arm in forced Y-maze. n = 8 mice per group. c Discrimination index in the novel object recognition test. n = 8 mice per group. d Western blotting of AT8, pThr231 and pSer416 in the hippocampal homogenates of Tau-CON mice and Tau-OVER mice. e Quantitation of the levels of AT8, pThr231 and pSer416 in (d). f Representative images of AT8 (cyan), pThr231 (red) and pSer416 (red) fluorescence staining in the hippocampal DG region of Tau-CON mice and Tau-OVER mice. Scale bars, 50 μm. g Quantification of AT8, pThr231 and pSer416 fluorescent areas in (f). n = 5 mice per group. h Western blotting of PP2A, p-CaMKII-α and CDK5 in the hippocampal homogenates of Tau-CON mice and Tau-OVER mice. i Quantitation of the levels of PP2A, p-CaMKII-α and CDK5 in (h). j Immunolabeling of PSD95 (red) and synaptophysin (SYN) (green) puncta in the brains of Tau-CON mice and Tau-OVER mice. Circles indicate Co-localization of PSD95 and SYN puncta. Scale bars, 5 μm. k Quantification of synaptic puncta or their apposition in (j). n = 5 mice per group. l Western blotting of PSD95 and SYN in the hippocampal homogenates of Tau-CON mice and Tau-OVER mice. m Quantitation of the levels of PSD95 and SYN in the brains of mice in (l). In d, h and l, data are representative of three independent experiments. In e, i and m, data are pooled from three independent experiments. Data are presented as mean ± SEM, and analyzed with unpaired t-test with two-tailed P values (a, c, e, g, i, k, m) or Mann–Whitney test (b). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: Meanwhile, at 48 h after infection, the cells were treated with 2.5 μM roscovitine [28] (MedChemExpress, #HY-30237) for 24 h to inhibit CDK5, or 5 μM DT061 [29] (MedChemExpress, #HY-112929) for 24 h to activate PP2A.

Techniques: Over Expression, Phospho-proteomics, Western Blot, Quantitation Assay, Fluorescence, Staining, Immunolabeling, Two Tailed Test, MANN-WHITNEY