dynamin inhibitor  (TargetMol)

Journal: Journal of Virology

Article Title: Bovine coronavirus enters HRT-18 cells via membrane fusion and clathrin-mediated endocytosis in a low pH-, dynamin-, cholesterol-, microtubule-, Rab7-, and Rab11-dependent manner

doi: 10.1128/jvi.01274-25

Figure Lengend Snippet: BCoV entry into HRT-18 cells depends on dynamin. ( A ) The maximum safe concentrations of dynasore were determined using the CCK-8 assay. ( B ) Western blot analysis was used to evaluate the BCoV N protein expression levels, with grayscale analysis performed and presented as a bar graph. ( C ) RT-qPCR was performed to assess the BCoV gene copy numbers. ( D ) TCID 50 assay was used to measure the BCoV viral titers in the cell supernatant. ( E ) IFA was used to detect the number of BCoV-infected cells. Scale bar = 100 µm. ( F ) RT-qPCR was used to evaluate the effect of dynasore on the viral entry. ( G ) RT-qPCR was used to evaluate the effect of dynasore on the viral attachment. ( H ) The siRNA silencing efficiency of dynamin was screened; the effects of dynamin-silenced cells on BCoV infection were assessed by ( I ) Western blot, ( J ) RT-qPCR, ( K ) TCID 50 , and ( L ) IFA. ( M ) RT-qPCR was used to evaluate the effect of dynamin-silenced cells on the viral entry; ( N ) RT-qPCR was used to evaluate the effects of dynamin-targeting siRNA on BCoV attachment. Data are presented as the mean ± SD of three independent experiments (not significant, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The inhibitors used in this study included SSAA09E3 (Cat HY-138102, MedChemExpress), a novel inhibitor that blocks the fusion of the viral membrane with the host cell membrane; CPZ (Cat C0982, Sigma), a clathrin-mediated endocytosis inhibitor; nystatin (Cat 475914, Sigma), a caveolae inhibitor that acts as a sterol-binding agent disrupting caveolae; blebbistatin (Cat 203391, Sigma), an inhibitor of micropinocytosis; dynasore (Cat T1848, TargetMol), a dynamin inhibitor; MβCD (Cat T4072, TargetMol), a cholesterol depletion inhibitor; chloroquine (Cat S6999, Selleck) and NH 4 Cl (Cat A9434, Sigma), a potent inhibitor of V-ATPase and a specific inhibitor of acidification of endosomal vesicles; colchicine (Cat HY-16569, MedChemExpress), which inhibits the polymerization of tubulin; E64d (Cat S7393, Selleck), a cathepsin inhibitor; and camostat (Cat HY-13512, MedChemExpress), a TMPRSS2 inhibitor.

Techniques: CCK-8 Assay, Western Blot, Expressing, Quantitative RT-PCR, Infection

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: Impact of activated DRP1 on mitophagy. (A, B) DCFH‐DA staining to assess ROS production in microglia of each group. Scale bar = 25 µm. (C, D) Fluorescence images and corresponding quantitative data displaying Mito‐ROS MitoSOX fluorescence in microglia of each group. Scale bar = 25 µm. (E, F) Observation of autophagic flux in microglia using the mRFP‐GFP‐LC3 reporter system. Scale bar = 15 µm. (G, H) Western blot analysis of DRP1 activation status. (I, J) DCFH‐DA staining to detect ROS production in microglia of each group. Scale bar = 25 µm. (K, L) Quantification of Mito‐ROS MitoSOX fluorescence in microglia of each group through fluorescence images. Scale bar = 25 µm. (M, N) Evaluation of autophagic flux in microglia utilizing the mRFP‐GFP‐LC3 reporter system. Scale bar = 15 µm. (O, P) Western blot results and quantification of mitochondrial‐specific proteins. All cellular experiments were repeated three times. * 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: Staining, Fluorescence, Western Blot, Activation Assay

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 activated DRP1 on microglia activation. (A) Immunohistochemical staining of microglia marker Iba‐1 in brain tissue, Scale bar = 200/50/10 µm. (B) Endpoint/branch length/density of microglia in each group. (C) H&E staining of the cerebral cortex post‐CIRI, scale bar = 50/25 µm. (D) Cellular body morphological changes in microglia of each group. Scale bar = 25 µm. (E) ELISA analysis of secretion levels of inflammatory factors TNF‐α, IL‐6, IL‐1β, and CCL2 in microglia of each group. Each group consist of six mice, and all cellular experiments were repeated three times. * 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: Activation Assay, Immunohistochemical staining, Staining, Marker, Enzyme-linked Immunosorbent Assay

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: