Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 1. Spinal cord injury induces upregulation of expression of E2F1 and its downstream targets. Analysis of expression of E2F1 and its transcriptional target genes in intact and injured spinal cord were performed by western blotting. A. 5-mm-long segment centered at the injury epicenter was dissected and homogenized in RIPA buffer. Equal amounts of protein were electrophoretically separated on NuPAGE Novex Bis-Tris gradient gels, transferred to nitrocellulose membranes, and blotted with antibodies to E2F1 and CDK1. GAPDH signal served as a loading control. B– C. The signal quantifications for E2F1 (B) and CDK1 (C) using Gel-Pro Analyzer software are displayed. E2F1 and CDK1 expression level was upregulated as early as 15 min and sustained until 3 days after injury. D–E. Cyclin A expression was increased at all time points. F. Bim and c-Myb, downstream targets of E2F1 were upregulated as early as 5 h post-injury and sustained until day 3 after SCI. G–H. Quantification of respective western blots in panel D. N = 4 rats/time point. *P,0.05 vs sham group. doi:10.1371/journal.pone.0042129.g001

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Expressing, Western Blot, Control, Software

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 2. CDK1 activity is increased after spinal cord injury. Western blotting analysis of common CDK substrates, CDK1 co-activator cyclin B1 and phosphorylations of specific CDK1 substrate (Ser54)-n-myc was performed in homogenates obtained from intact and injured spinal cord. A. Cyclin B1 expression was upregulated at all time points tested. Phosphorylation (Ser54) of n-myc and phospho-CDK substrate motif signal levels were increased from 5 h to day 7. B–D. Quantification of respective western blots in panel A. n = 4 rats/time point. *P,0.05 vs sham group. doi:10.1371/journal.pone.0042129.g002

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Activity Assay, Western Blot, Expressing, Phospho-proteomics

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 3. The temporal profile and cell specificity of E2F1 and CDK1 expression after SCI. A–B. Coronal section in intact spinal cord (A) showed that E2F1 is relatively weak and detected mainly in neurons of the gray matter. At 24 h after SCI, E2F1 immunoreactivity was upregulated not only in gray matter but also in lesion area (B). C. E2F1+ cells were also co-labelled with NeuN in the dorsal horn of the gray matter at 1 day after SCI. D–E. Only a small subset of E2F1+ cells in the lesion area were positive for OX42 at 24 h (D) and 7 d (E) after SCI. F–G. In the intact spinal cord (F), CDK1 immunoreactivity is relatively weak and detected mainly in motor neurons in the ventral horn and CC1+ oligodendrocytes. At 24 h after SCI (G), CDK1 immunoreactivity was upregulated not only in the ventral horn but also in the spared white matter, colocalized with CC1+ oligodendrocytes. CDK1+ cells also appeared in the lesion area. H–I. CDK1 was expressed by CC1+ oligodendrocytes in the white matter in the intact spinal cord (H) and at 1 day after SCI. J. Only a small subset of CDK1+ cells in the lesion area were positive for OX42 at 24 h after SCI. K. Coronal section in intact spinal cord (a) shows that E2F1 was expressed in the motor neurons in the ventral horn. Immunoreactivity of E2F1 (b–d) was increased at 5 h, and 1–3 days post injury, and highly expressed by motor neurons. L. In intact spinal cord (a), CDK1/NeuN was detected in the motor neurons in the ventral horn. At 5 h after injury, immunoreactivity of CDK1 (b) was increased and sustained until 3 days post injury (c–d), and highly expressed by motor neurons. All images were taken at 2 mm rostral to epicenter. Scale bar = 500 mm for A–B, F–G. Scale bar = 100 mm for C–E, H–J, K–L. doi:10.1371/journal.pone.0042129.g003

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Expressing

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 4. E2F1 gene silencing down-regulates endogenous CDK1 expression in vitro. A. 27 mer siRNA duplexes for human E2F1 or trilencer-27 universal scrambled negative control siRNA duplex was transfected in the human neuroblastoma SH-SY5Y cells. Two days after transfection, the cells were harvested and subjected to western blotting using mouse monoclonal antibodies to E2F1 and CDK1. Transfection with shRNA against E2F1 resulted in reduction of E2F1 expression (58% to 66% of control), accompanied by 50% of reduction of CDK1 expression. B. Primary rat cerebral cortical neurons were transfected with shRNA against rat E2F1. E2F1 protein expression was robust reduced to 47% or 59% for shRNAs 1 and 2 respectively, and E2F1 knockdown resulted in reduction of CDK1 expression from 47% to 64% for shRNAs 1 and 2 respectively. N = 4 dishes from 3 independent culture. *P,0.05 vs sham group. doi:10.1371/journal.pone.0042129.g004

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Expressing, In Vitro, Negative Control, Transfection, Western Blot, Bioprocessing, shRNA, Control, Knockdown

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 5. Colocalization of E2F1/CDK1 upregulation with neuronal apoptosis in injured spinal cord. A–C. Western blot analysis shows a significant increase in biochemical markers of apoptosis, active caspase-3 signal, as well as 145/150 kDa cleavage product of a-fodrin after SCI. N = 4 rats/time points. *p,0.05 vs sham group. D. E2F1+ cells were co-label with cleaved caspase 3 (yellow, arrow heads) in the gray matter at 2 mm rostral to the epicenter at 1 day after SCI. Scale bar = 100 mm. E. Coronal section in intact spinal cord (top panel) shows that CDK1 was expressed in the motor neurons in the ventral horn (VH). At 1 day after injury, immunoreactivity of CDK1 (middle panel, green) was increased, and highly expressed by apoptotic motor neurons (red), as shown at 2 mm rostral to epicenter. CDK1 was rarely expressed by inter-neurons in the dorsal horn (DH) after SCI (bottom panel). Scale bar = 100 mm for D(a–h) and 500 mm for D(i–l). doi:10.1371/journal.pone.0042129.g005

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Western Blot

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 6. E2F1/CDK1 expression is necessary for trophic deprivation-induced neuronal apoptosis. Rat cortical neurons were co- transfected with expression plasmids for ß-galactosidase with, either empty vector or vector expressing E2F1 or CDK1 (A and B). Similarly, ß- galactosidase plasmid was co-transfected along with scrambled, E2F1 or CDK1 shRNAs (C, D and E) and the extent of apoptosis was examined 48 h after transfection, or after an additional 24 h of trophic deprivation (TD) induction post 48 h transfection. A. Neurons transfected with E2F1 vector (0.6 mg DNA/0.56106 neurons) increased basal apoptosis as compared to empty vector. B. Neurons transfected with CDK1 (0.8 mg DNA/0.56106

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 7. Pharmacological inhibition of CDK1 blocks neuronal apoptosis. Cortical neurons were pre-treated with Roscovitine or CR8 (CDK1 inhibitors) or vehicle and then exposed to TD-or campthotecin induced apoptosis. A. Representative photomicrographs of control and trophic deprived neurons treated with the indicated concentrations Roscovitine and CR8 are shown. Upper row presents phase contrast images (Healthy neurons are indicated by larger cell bodies and abundant processes; Apoptotic neurons display shrunken cell bodies and sparse or lost processes). Lower row shows chromatin staining with Hoechst 33258. Arrows and arrowheads indicate surviving and apoptotic neurons, respectively suggesting an attenuation of TD-induced neuronal death in neurons pre-treated with Roscovinine or CR8. B. A quantitative assessment of the percentage of nuclei featuring chromatin condensation demonstrates a significant attenuation of TD-induced apoptosis in neurons pre-treated with Roscovitine (10 mM; *p,0.05, vs. TD vehicle) whereas CR8 at concentrations as low as 1 mM (***p,0.001, vs. TD vehicle) almost completely blocked development of apoptotic features in neuronal nuclei. C. Significant attenuation of campthotecin-induced apoptosis in neurons pre-treated with Roscovitine (50 mM; *p,0.001, vs. vehicle) and CR8 at concentrations as low as 1 mM (***p,0.001, vs. vehicle). doi:10.1371/journal.pone.0042129.g007

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Inhibition, Control, Staining

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 8. CR8 administration reduces SCI-induced activation of the E2F1/CDK1 signaling pathway. Samples were obtained from rats exposed to spinal cord injury and CR8 treatment (1 mg/kg intraperitoneal administration) and analyzed by western blotting. Equal protein loading is demonstrated by consistent GAPDH levels. A. CR8 attenuated SCI mediated increase in E2F1 and its target cyclin A expression. B–C. Quantification of respective western blots in panel A. D. CR8 reduced SCI induced increase in phospho-(Ser54)-n-myc, phosphorylated CDK substrates and expression of cyclin B1. E–G. Quantification of respective western blots in panel E. H–J. Administration of CR8 significantly reduced Bim and c-Myb expression at 24 h after SCI. H shows representative Western blots for Bim, c-Myb, and the loading control, GAPDH. I and J show quantitative analysis of Bim and c- Myb expression. N = 4. *p,0.05 vs. vehicle group. doi:10.1371/journal.pone.0042129.g008

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques: Activation Assay, Western Blot, Expressing, Control

Journal: PloS one

Article Title: Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

doi: 10.1371/journal.pone.0042129

Figure Lengend Snippet: Figure 9. SCI-induced immunoreactivity of E2F1 and CDK1 was attenuated by CR8 treatment. A. Coronal section in intact spinal cord (a– c) shows that E2F1 was expressed in the motor neurons in the ventral horn. At 1 day after injury, immunoreactivity of E2F1 (d–f) was increased, and highly expressed by motor neurons. The upregulation of E2F1 was clearly attenuated by CR8 treatment (g–i). B. In intact spinal cord (a–c), CDK1/ NeuN was detected in the motor neurons in the ventral horn. At 1 day after injury, immunoreactivity of CDK1 (d–f) was increased, and highly expressed by motor neurons. CDK1 upregulation was attenuated by CR8 treatment (g–i). All images were taken at 2 mm rostral to epicenter. Scale bar = 100 mm for C–F. doi:10.1371/journal.pone.0042129.g009

Article Snippet: 27 mer siRNA duplexes for human E2F1 (ID 1869), trilencer-27 universal scrambled negative control siRNA duplex (SR30004), constructs expressing 29 mer shRNAs against Rat Cdc2 and E2F1 in pGFPV-RS vectors were obtained from Origene Technologies Inc. Rat Neuron NucleofectorH Kit (VPG-1003) was purchased from Lonza.

Techniques:

Journal: bioRxiv

Article Title: NF-κB-Dependent Transcriptional Regulation of Piezo1 Mediates Bacterial Clearance on Stiffened Lung Matrix

doi: 10.1101/2025.11.06.687026

Figure Lengend Snippet: WT, Piezo1 fl/fl , and Piezo1 LysMCre BMDMs were treated with heat-inactivated and live P. aeruginosa (green). Piezo1 Ca 2+ channel activity, phagolysosome maturation, and bacterial clearance were measured on polyacrylamide gels of pathophysiologic range lung stiffness (1 kPa: normal lung, 8-25 kPa: injured lung) and standard tissue culture conditions (10 kPa). ( A ) Phagolysosome maturation was measured using pH-sensitive fluorescent pHrodo bioparticles in P. aeruginosa -treated WT BMDMs on pathophysiologic-range matrix stiffness (1, 8, 25 kPa), n = 5. Phagolysosome maturation is stiffness-dependent and requires matrix stiffness which resembles injured lung (25 kPa). Phagolysosome maturation is reduced upon downregulation of Piezo1 by ( B ) Cre recombinase, as quantified in ( C ), or ( D ) siRNA, as quantified in ( E ), n = 3. ( F ) Bacterial clearance was measured using colony forming units (CFU) of P. aeruginosa . Bacterial clearance is enhanced in Piezo1-sufficient (Piezo1 fl/fl ) BMDMs, n= 3. Data are presented as mean ± SEM. Comparisons by one-way ANOVA or t-test, as appropriate. Scale bars = 25 µm. Images were taken at 40x original magnification. p-values are as indicated.

Article Snippet: BMDMs were given Piezo1-targeted (Origene, Cat. No. SR423525) or control siRNA (Origene, Cat. No. SR30005) and treated with nucleofection solution (Lonza, Cat. No. VCA-1003) prior to nucleofection with program D-032.

Techniques: Activity Assay