Journal: EMBO Reports

Article Title: TMPRSS2-induced Golgi disruption restricts the incorporation of virus envelope glycoproteins into virions

doi: 10.1038/s44319-026-00797-2

Figure Lengend Snippet: ( A ) Phosphorylated and total ERK (pERK and ERK, respectively) were detected in 293 T cells expressing CD4-mCherry (Control), TMPRSS2-mCherry (TM2-WT), TMPRSS2-dCT-mCherry (TM2-dCT), or TMPRSS2-S441A-mCherry (TM2-S441A) at the indicated time (h) post-transfection by immunoblotting analysis. The ratio of pERK to total ERK at 0 hr was set as 1, and the ratio of pERK to total ERK at each time point to that is shown in the graph (the quantified results are expressed as mean ± SEM of three separate experiments, n = 3). The samples were obtained from the same experiment, and the gels and blots were processed in parallel. The results of Student’s t tests for the value of Ctrl at each time point are shown above the points. ( B ) IPA analysis of differentially expressed proteins between TM2-WT- and TM2-S441A-transfected 293T cells. ( C ) Experimental scheme for ( D , E ). ( D ) Inhibition of TM2-induced Golgi disruption using ERK or CDK inhibitors was analyzed in 293T cells, transfected with Golgi-YFP (green) and TM2-WT (red), and cultured for 24 h in the presence of 200 μM nafamostat. After removing nafamostat, the cells were cultured with the indicated concentrations of ERK or CDK5 inhibitor for 180 min before undergoing fixation. ( E ) Quantitative analysis of the images. Sixty cells were randomly selected from at least three independent images and plotted. Representative data from three independent experiments are shown. Scale bars indicate 50 μm. The results of Student’s t tests are shown above the bars. .

Article Snippet: CDK5 inhibitor 20-233 , MCE , HY-123772.

Techniques: Expressing, Control, Transfection, Western Blot, Inhibition, Disruption, Cell Culture

Journal: EMBO Reports

Article Title: TMPRSS2-induced Golgi disruption restricts the incorporation of virus envelope glycoproteins into virions

doi: 10.1038/s44319-026-00797-2

Figure Lengend Snippet: ( A ) Inhibition of TMPRSS2-induced Golgi disruption using ERK or CDK inhibitors, analyzed in 293T cells, transfected with Golgi-YFP (green) and TMPRSS2-WT-mCherry (TM2-WT-Cherry, red) and cultured for 24 h with 200 μM nafamostat. After removing nafamostat, cells were cultured with 20 μM ERK (20-233) or CDK1 (Ro 3306) inhibitors for 180 min before fixation. Scale bars indicate 10 μm. ( B ) Quantitative analysis of Golgi disruption in the images. Forty cells were randomly selected from at least three independent images and plotted. Representative data from two independent experiments are shown. ( C ) Immunoblotting analysis of CoV-2-S pseudotyped lentiviruses in 293T cells expressing CD4 (Ctrl), or TMPRSS2 (TM2) with or without 50 μM of ERK inhibitor. The ratio of CoV-2-S2 to p24 was calculated based on band density (right, the quantified results are expressed as mean ± SEM of three separate experiments, n = 3). ( D ) Inhibition of TM2 function by CoV-2-E. Luciferase activity for the infectivity of CoV-2-S pseudotyped lentiviruses produced in CD4-mCherry- (CD4)-, or TMPRSS2-mCherry- (TM2)-, and CoV-2-E or CoV-2-EdN14 expressing 293 T cells (the quantified results from a representative experiment in the two experiments independently conducted are expressed as mean ± SEM, n = 6). ( E ) Immunofluorescence analysis of Golgi-YFP (green) and CoV-2-E (blue) in CD4-mCherry (CD4-Cherry)-, TMPRSS2-WT-mCherry (TM2-WT-Cherry)-, TMPRSS2-dCT-mCherry (TM2-dCT-Cherry)-, or TMPRSS2-S441A-mCherry (TM2-S441A-Cherry)-expressing 293T cells cultured with CoV-2-E inhibitory peptide (+) or control peptide (−) at 24 h post-transfection. ( F ) Immunofluorescence analysis for CoV-2-S structural changes using anti-CoV-2-S2 (1A9, green) antibodies in CoV-2-S and CoV-2-E (blue) in CD4-Cherry-, TM2-WT-Cherry, TM2-dCT-Cherry, or TM2-S441A-Cherry (red) -expressing 293 T cells cultured with CoV-2-E inhibitory peptide (+) or control peptide (−) at 24 h post-transfection. Scale bars indicate 50 μm. The results of Student’s t tests are shown above the bars.

Article Snippet: CDK5 inhibitor 20-233 , MCE , HY-123772.

Techniques: Inhibition, Disruption, Transfection, Cell Culture, Western Blot, Expressing, Luciferase, Activity Assay, Infection, Produced, Immunofluorescence, Control

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: Phospho-proteomics, 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: