Journal: Scientific Reports

Article Title: CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression

doi: 10.1038/srep02932

Figure Lengend Snippet: (a) immunoblots of CDK5 and p35 protein expression in MCF10A mammary epithelial cells and breast cancer cells. (b) immunoblotting analysis of CDK5 and p35 protein expression in breast cancer tissue specimens, including non-cancerous surrounding tissues (N) and cancerous tissues (Ca). (c) immunohistochemistry of CDK5 protein in breast cancer tissue specimens. Left , weak staining; middle , moderate staining; right , strong staining. Scale bar = 100 μm. (d), (e) and (f) percentages of human breast cancer specimens with high level of CDK5 expression in different tumor subtypes and different tumor grades. Corresponding p -values analyzed by χ2 test are indicated.

Article Snippet: Antibodies against the following proteins were used: CDK5 (1:500), p35 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FAK (1:1000), phosphorylated FAK Tyr-397 (1:1000), Smad2/3 (1:1000), p-Smad2 (1:1000) (Cell Signaling Technology), E-cadherin (1:2000), N-cadherin (1:2000), Vimentin (1:6000), Fibronectin (1:5000) (BD Transduction Laboratories, Lexington, KY, USA), Occludin (1:2000) (Invitrogen, Carlsbad, CA,USA), α-SMA (1:4000) (Sigma), phosphorylated FAK Ser-732 (1:1000), GAPDH (1:5000) (Millipore, Billerica, MA, USA) and GFP (1:2000) (Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression

doi: 10.1038/srep02932

Figure Lengend Snippet: Correlation of CDK5 expression with breast tumor subtypes

Article Snippet: Antibodies against the following proteins were used: CDK5 (1:500), p35 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FAK (1:1000), phosphorylated FAK Tyr-397 (1:1000), Smad2/3 (1:1000), p-Smad2 (1:1000) (Cell Signaling Technology), E-cadherin (1:2000), N-cadherin (1:2000), Vimentin (1:6000), Fibronectin (1:5000) (BD Transduction Laboratories, Lexington, KY, USA), Occludin (1:2000) (Invitrogen, Carlsbad, CA,USA), α-SMA (1:4000) (Sigma), phosphorylated FAK Ser-732 (1:1000), GAPDH (1:5000) (Millipore, Billerica, MA, USA) and GFP (1:2000) (Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Scientific Reports

Article Title: CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression

doi: 10.1038/srep02932

Figure Lengend Snippet: (a) morphologic change of MCF10A cells cultured without or with TGF-β1 (5 ng/ml, 48 h), Scale bar = 100 μm. (b) immunoblotting analysis of expression of CDK5 and the epithelial marker E-cadherin, and the mesenchymal markers N-cadherin and α-SMA. (c) immunofluorescence staining for the epithelial marker E-cadherin, and the mesenchymal markers N-cadherin and α-SMA. Scale bar = 50 μm. (d) immunoblotting analysis of CDK5 and p35 protein expression in MCF10A cells without or with TGF-β1 induction and its inhibitor LY364947. The smad2/3 and p-smad2 were used as the positive control of cultured with TGF-β1. (e), (f), (g) and (h) real-time PCR analysis of CDK5 and p35 mRNA expression upon TGF-β1 treatment. Error bars represent the mean ± SD of triplicate experiments.

Article Snippet: Antibodies against the following proteins were used: CDK5 (1:500), p35 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FAK (1:1000), phosphorylated FAK Tyr-397 (1:1000), Smad2/3 (1:1000), p-Smad2 (1:1000) (Cell Signaling Technology), E-cadherin (1:2000), N-cadherin (1:2000), Vimentin (1:6000), Fibronectin (1:5000) (BD Transduction Laboratories, Lexington, KY, USA), Occludin (1:2000) (Invitrogen, Carlsbad, CA,USA), α-SMA (1:4000) (Sigma), phosphorylated FAK Ser-732 (1:1000), GAPDH (1:5000) (Millipore, Billerica, MA, USA) and GFP (1:2000) (Abcam, Cambridge, UK).

Techniques: Cell Culture, Western Blot, Expressing, Marker, Immunofluorescence, Staining, Positive Control, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression

doi: 10.1038/srep02932

Figure Lengend Snippet: (a) and (b) assessment of the repression efficiency of CDK5 mRNA (a) and protein (b) expression after retroviral infection in MCF10A cells. Error bars represent the mean ± SD of triplicate experiments. (c) morphologic change of MCF10A cells in culture with TGF-β1 after infection of shCDK5- 1# or empty vector, Scale bar = 200 μm. (d) immunoblotting analysis of expression of the epithelial marker E-cadherin and the mesenchymal markers N-cadherin and α-SMA in MCF10A cultured without or with TGF-β1 after infection of shCDK5- 1# or empty vector. (e) immunofluorescence staining for the epithelial marker E-cadherin and mesenchymal marker N-cadherin. Scale bar = 50 μm.

Article Snippet: Antibodies against the following proteins were used: CDK5 (1:500), p35 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FAK (1:1000), phosphorylated FAK Tyr-397 (1:1000), Smad2/3 (1:1000), p-Smad2 (1:1000) (Cell Signaling Technology), E-cadherin (1:2000), N-cadherin (1:2000), Vimentin (1:6000), Fibronectin (1:5000) (BD Transduction Laboratories, Lexington, KY, USA), Occludin (1:2000) (Invitrogen, Carlsbad, CA,USA), α-SMA (1:4000) (Sigma), phosphorylated FAK Ser-732 (1:1000), GAPDH (1:5000) (Millipore, Billerica, MA, USA) and GFP (1:2000) (Abcam, Cambridge, UK).

Techniques: Expressing, Retroviral, Infection, Plasmid Preparation, Western Blot, Marker, Cell Culture, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression

doi: 10.1038/srep02932

Figure Lengend Snippet: (a) immunoblotting analysis of expression of CDK5 and p35, the epithelial marker E-cadherin, and the mesenchymal markers N-cadherin and α-SMA in MCF10A-Vector, MCF10A-CDK5, MCF10A-p35 and MCF10A-CDK5-p35 cells. (b) immunofluorescence staining for the mesenchymal marker α-SMA. Scale bar = 50 μm. (c) immunoblotting analysis of expression of CDK5, the epithelial marker E-cadherin, the mesenchymal markers α-SMA, FAK, p-FAK Y397 and p-FAK S732 in MCF10A-Vector, MCF10A-CDK5 and MCF10A-CDK5dn cells with or without TGF-β1 treatment. (d) and (e) migration (24 h; d) and invasion (60 h; e) assays in MCF10A-Vector, MCF10A-CDK5 and MCF10A-CDK5dn cells with or without TGF-β1 treatment. The mean was derived from cell counts of 5 fields, and each experiment was repeated 3 times (***, P < 0.001, compared with the control). Representative images of migrated and invaded cells are shown ( upper ).

Article Snippet: Antibodies against the following proteins were used: CDK5 (1:500), p35 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FAK (1:1000), phosphorylated FAK Tyr-397 (1:1000), Smad2/3 (1:1000), p-Smad2 (1:1000) (Cell Signaling Technology), E-cadherin (1:2000), N-cadherin (1:2000), Vimentin (1:6000), Fibronectin (1:5000) (BD Transduction Laboratories, Lexington, KY, USA), Occludin (1:2000) (Invitrogen, Carlsbad, CA,USA), α-SMA (1:4000) (Sigma), phosphorylated FAK Ser-732 (1:1000), GAPDH (1:5000) (Millipore, Billerica, MA, USA) and GFP (1:2000) (Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Marker, Plasmid Preparation, Immunofluorescence, Staining, Migration, Derivative Assay, Control

Journal: Scientific Reports

Article Title: CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression

doi: 10.1038/srep02932

Figure Lengend Snippet: (a) and (b) migration (24 h; a) and invasion (48 h; b) assays in MDA-MB-231 and BT549 cells after infection of shCDK5- 1# or empty vector. The mean was derived from cell counts of 5 fields, and each experiment was repeated 3 times (***, P < 0.001, compared with the control). Representative images of migrated and invaded cells are shown. (c) immunoblotting analysis of expression of CDK5 protein, and the mesenchymal marker α-SMA in MDA-MB-231 and BT549 cells after infection of shCDK5- 1# or empty vector. (d) tumors from the BALB/c female nude mice that were subcutaneously injected with MDA-MB-231 and BT549 cells stably expressing shCDK5- 1# . (e) weight of tumors at day 60 after injection. (***, P < 0.001, compared with the control). Representative images of migrated and invaded cells are shown.

Article Snippet: Antibodies against the following proteins were used: CDK5 (1:500), p35 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FAK (1:1000), phosphorylated FAK Tyr-397 (1:1000), Smad2/3 (1:1000), p-Smad2 (1:1000) (Cell Signaling Technology), E-cadherin (1:2000), N-cadherin (1:2000), Vimentin (1:6000), Fibronectin (1:5000) (BD Transduction Laboratories, Lexington, KY, USA), Occludin (1:2000) (Invitrogen, Carlsbad, CA,USA), α-SMA (1:4000) (Sigma), phosphorylated FAK Ser-732 (1:1000), GAPDH (1:5000) (Millipore, Billerica, MA, USA) and GFP (1:2000) (Abcam, Cambridge, UK).

Techniques: Migration, Infection, Plasmid Preparation, Derivative Assay, Control, Western Blot, Expressing, Marker, Injection, Stable Transfection

Journal: Scientific Reports

Article Title: CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression

doi: 10.1038/srep02932

Figure Lengend Snippet: (a) immunoblotting analysis of expression of FAK and p-FAK S732 in MCF10A cells cultured without or with TGF-β1 after infection of shCDK5- 1# or empty vector. (b) and (c) immunoblotting analysis of expression of CDK5, FAK, p-FAK Y397 and p-FAK S732 in MDA-MB-231 (b) and BT549 (c) cells after infection of shCDK5- 1# or empty vector.

Article Snippet: Antibodies against the following proteins were used: CDK5 (1:500), p35 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FAK (1:1000), phosphorylated FAK Tyr-397 (1:1000), Smad2/3 (1:1000), p-Smad2 (1:1000) (Cell Signaling Technology), E-cadherin (1:2000), N-cadherin (1:2000), Vimentin (1:6000), Fibronectin (1:5000) (BD Transduction Laboratories, Lexington, KY, USA), Occludin (1:2000) (Invitrogen, Carlsbad, CA,USA), α-SMA (1:4000) (Sigma), phosphorylated FAK Ser-732 (1:1000), GAPDH (1:5000) (Millipore, Billerica, MA, USA) and GFP (1:2000) (Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Cell Culture, Infection, Plasmid Preparation

Journal: Scientific Reports

Article Title: CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression

doi: 10.1038/srep02932

Figure Lengend Snippet: (a) Co-IP of CDK5, FAK and p35 in 293T cells transfected with CDK5 and FAK-GFP. (b) Co-IP of endogenous CDK5, FAK and p35 in MCF10A cells cultured without or with TGF-β1.

Article Snippet: Antibodies against the following proteins were used: CDK5 (1:500), p35 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FAK (1:1000), phosphorylated FAK Tyr-397 (1:1000), Smad2/3 (1:1000), p-Smad2 (1:1000) (Cell Signaling Technology), E-cadherin (1:2000), N-cadherin (1:2000), Vimentin (1:6000), Fibronectin (1:5000) (BD Transduction Laboratories, Lexington, KY, USA), Occludin (1:2000) (Invitrogen, Carlsbad, CA,USA), α-SMA (1:4000) (Sigma), phosphorylated FAK Ser-732 (1:1000), GAPDH (1:5000) (Millipore, Billerica, MA, USA) and GFP (1:2000) (Abcam, Cambridge, UK).

Techniques: Co-Immunoprecipitation Assay, Transfection, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

doi: 10.1074/jbc.m109.064311

Figure Lengend Snippet: FIGURE 7. Suppression of JCV genome replication by abrogation of G2 arrest. Effect of inhibition of G2 check- point signaling on JCV DNA replication as determined by the DpnI replication assay. IMR-32 cells transfected with pBS-JCori and an expression vector for TAg were incubated for 24 h before addition of dimethyl sulfoxide (DMSO) (control), 50 nM ucn-01, or 2.5 mM caffeine (caf). The siRNA against non-targeting control (Ct) or Wee1 or/and Cdc2 were transfected 24 h before transfection of the plasmids. The collected cells were subjected to immunoblot anal- ysis with antibodies to FLAG-TAg, Wee1, and Cdc2 (A), flow cytometric analysis of cell cycle profile for the TAg cell subset (B), and DpnI replication assay (C and D). Replicated DNA extracted from the cells was detected by Southern blotanalysiswithaDNAprobespecificforpBS-JCori(C).TheintensityofDNAbandswasquantifiedandisindicated inthebargraphrelativetothevalueofdimethylsulfoxide(forucn-01andcaffeine)orthatofsiCt(forsiWee1,siCdc2, and siWeesiCdc2) (D); data are mean S.D. of triplicates from a representative experiment.

Article Snippet: Antibodies to Chk1, Ser317-phosphorylated Chk1, Chk2, Thr68-phosphorylated Chk2, Tyr15-phosphorylated Cdc2, and Ser15-phosphorylated p53 were obtained from Cell Signaling Technology (Beverly,MA).

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Incubation, Control, Western Blot, FLAG-tag

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: A. Single nucleus RNAseq data from hDRG showing the expression of CDK5 and CDK5R1 (encoding p35). Data are derived from Nguyen et al., 2021 . B. A human p35 expression vector was transfected into N2a cells. An empty vector (EV) and a His-tagged p35 vector were used as controls. Western blot for p35 was performed before sending the expression vector to be packaged into HSV. A 35 kDa band corresponding to the untagged human p35 was detected by Western. C. SH-SY5Y cells were differentiated and then transfected with a His-tagged p35 expression vector or an empty vector. Next, cells were treated with the following inflammatory mediators: Lane 1, non-treated; lane 2, 100nM bradykinin (BK) + 1µM PGE 2 (PG); lane 3, 10µM BK + 10µM PG; lane 4, 100nM BK + 1µM PG + 10µM 5HT + 10µM histamine; lane 5, 100nM BK + 1µM PG + 1mM 5HT + 1mM histamine; lane 6, 10µM BK + 10µM PG + 10µM 5HT + 10µM histamine; lane 7, 10µM BK + 10µM PG + 1mM 5HT + 1mM histamine. CDK5/p35 was immunoprecipitated and kinase activity was evaluated by the level of phosphorylated P histone H1, a substrate of cyclin-dependent kinases.

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques: Expressing, Derivative Assay, Plasmid Preparation, Transfection, Western Blot, Immunoprecipitation, Activity Assay

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: A. Representative image showing the patched hDRG neurons under the inverted microscope. B. A representative AP trace ( black ) evoked at rheobase and a superimposed subthreshold trace without AP ( dashed orange trace ) in three groups (uninfected control; UI), (GFP control; GFP) and (p35 transfected; p35 hDRG neurons) before (baseline) and after perfusion with prostaglandin E2 (1μM) and bradykinin (100nM) ( red trace ). C. A representative enlarged AP trace depicting AP measurements. Rheobase: Minimal required threshold current to evoke an AP by an incrementing series of depolarizing pulse (incremental step; 50 pA and duration; 20 milliseconds), AP voltage threshold and resting membrane potential (RMP) are shown as dashed pink line and dashed red line respectively. AP rise time : AP rise to the peak, AP amplitude : AP peak height from RMP, AP overshoot: AP amplitude from zero membrane potential, AP fall time: AP peak back to the threshold value, AHP : (afterhyperpolarization) amplitude peak amplitude RMP. AP half width : AP duration at half the AP amplitude.

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques: Inverted Microscopy, Control, Transfection, Membrane

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: Bar diagrams (mean ± SEM) showing RMP (A), and rheobase current (B) in neurons from UI ( n=10; black circle ), GFP ( n=9; green circle ) and p35 ( n=9; orange circle ) groups. Statistical comparisons were performed using Kruskal Wallis test followed by Dunn’s multiple comparisons test. * p < 0.05, ns (non-significant).

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques:

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: Bar diagrams (mean ± SEM) showing AP rise time (A), AP fall time (B), AP half width (C), AP amplitude (D), AP overshoot (E), and AHP (F) in neurons from UI ( n=10; black circle ), GFP ( n=9; green circle ) and p35 ( n=9; orange circle ) groups. Statistical comparisons were performed using Kruskal Wallis test followed by Dunn’s multiple comparisons test. * p < 0.05, ** p < 0.01, ns (non-significant).

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques:

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: Paired dot plot graph depicting (A) RMP, and (B) rheobase current before (baseline; B) and after 1μM prostaglandin E2 and 100 nM bradykinin (PG/BK) in UI ( n=10; black circle ), GFP ( n=9; green circle ) and p35 ( n=9; orange circle ) neurons. In each section, the upper panel represents absolute value and lower panel represents % change. Statistical comparisons were performed using Wilcoxon signed rank test for paired data analysis and Kruskal Wallis test for percent changes. Dunn’s multiple comparisons test was performed for pairwise comparisons of percent changes. * p < 0.05, ** p < 0.01, ns (non-significant).

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques:

Journal: bioRxiv

Article Title: ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

doi: 10.1101/2023.05.31.543017

Figure Lengend Snippet: Paired dot plot graph depicting (A) AP rise time, (B) AP fall time, (C) AP half width, (D) AP amplitude, (E) AP overshot, (F) AHP before (baseline; B) and after 1μM prostaglandin E2 and 100 nM bradykinin (PG/BK) in UI ( n=10; black circle ), GFP ( n=9; green circle ) and p35 ( n=9; orange circle ) neurons. In each section, the upper panel represents absolute value and lower panel represents % change. Statistical comparisons were performed using Wilcoxon signed rank test for paired data analysis and Kruskal Wallis test for percent changes. Dunn’s multiple comparisons test was performed for pairwise comparisons of percent changes. * p < 0.05, ** p < 0.01, ns (non-significant).

Article Snippet: To confirm expression of the Cdk5 activator p35 before packaging into HSV, an expression vector with an untagged human p35, pCMV-p35 (gift from Dr. Li-Huei Tsai, Addgene plasmid # 1347, Watertown, MA), was transfected into Neuro 2a cells using Neuro-2a Cell Avalanchetransfection reagent (EZ Biosystems, College Park, MD).

Techniques: