Journal: iScience

Article Title: Blockage of lamin-A/C loss diminishes the pro-inflammatory macrophage response

doi: 10.1016/j.isci.2022.105528

Figure Lengend Snippet: Pro-inflammatory macrophage activation induces Lamin-A/C phosphorylation and degradation (A) Immunoblot shows total levels of Lamin-A and Lamin-C in Untreated (UT), and LPS-treated BMDMs. α-tubulin served as a loading control. (B) Time course quantifications of Lamin-A and Lamin-C normalized with α-tubulin over three independent immunoblotting experiments. Calculated values were further normalized to the UT BMDM condition to find the fold change. Data are presented as Mean ± S.E. (C) Longer exposure of immunoblots against Lamin-A/C in UT, and LPS-treated BMDMs to detect lower fragmented Lamin-A/C bands (50kDa and 40kDa). α-tubulin served as a loading control. (D) Box plots show degraded Lamin-A/C fragments (50kDa and 40kDa) normalized to α-tubulin between UT and LPS-treated BMDMs. Calculated values were further normalized to the UT BMDM condition to find the fold change. (E) Immunoblots against Lamin-A/C in UT, LPS-treated, LPS Wash-out BMDMs to detect fragmented Lamin-A/C bands (∼50kDa and ∼40kDa). α-tubulin served as a loading control. (F) Immunoblot shows total levels of p-(Ser22)-Lamin-A and p-(Ser22)-Lamin-C along with a phosphorylated fragmented band of Lamin-A/C (∼28kDa) in UT, and LPS-treated BMDMs. α-tubulin served as a loading control. (G) Time course quantification of total p-(Ser22)-Lamin-A+C normalized to the total Lamin-A/C over three independent immunoblotting experiments. Calculated values were further normalized to the UT BMDM condition to find the fold change. (H) (Top) Schematic shows the direct or indirect CDK1-mediated Lamin-A/C phosphorylation, which is known from other cells to be blocked by PP2A during the cell cycle. Also shown are the drugs used in this study to selectively inhibit the activity of the respective enzymes. (Bottom) Schematic shows Caspase-6 mediated Lamin-A/C degradation as inhibited by Z-VEID-FMK. (I) Box plots show the lowering of pro-inflammatory gene expression (qPCR) (Left) and secreted cytokine levels (ELISA) (Right) in LPS-treated BMDMs, also treated with RO-3306 or Z-VEID-FMK to inhibit Lamin-A/C phosphorylation and degradation, respectively. Levels were normalized to the 6 h LPS-treated BMDM condition to find the fold change. (J) Box plots show an increase in the pro-inflammatory gene expression (qPCR) (Left) and secreted cytokine levels (ELISA) (Right) in LPS-treated BMDMs, also treated with LB-100, a PP2A inhibitor, as compared to BMDMs treated with only LPS for 24 h. Levels were normalized to the 24 h LPS-treated BMDM condition to find the fold change. For all the plots p-values were obtained with the two-sided Student’s t -test. In all the box plots, the boxes show 25th and 75th percentiles, the middle horizontal lines show the median, small open squares show the mean, and whiskers indicate S.D. All the experiments were independently repeated three or more times.

Article Snippet: Rabbit polyclonal anti-phospho-CDK1 (Thr161) antibody , Abcam , Cat#ab194874.

Techniques: Activation Assay, Western Blot, Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Blockage of lamin-A/C loss diminishes the pro-inflammatory macrophage response

doi: 10.1016/j.isci.2022.105528

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho-CDK1 (Thr161) antibody , Abcam , Cat#ab194874.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, In Vitro, Derivative Assay, Software

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: RNAi duplex sequences

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: ISL1 interacts with CDK1. a–c Co-IP assays were performed to measure the ISL1/CDK1/cyclinB1 complex in MGC803 cells. Lysate was immunoprecipitated with antibodies against ISL1, CDK1, or cyclinB1 and analyzed by immunoblotting. IgG: immunoglobulin G. d Full-length or truncated ISL1 was used to construct GST-fusion proteins (Top) for pull-down assays with CDK1 protein (Bottom)

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Construct

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: ISL1 is serine-phosphorylated. a MGC803 cells lysates were immunoprecipitated (IP) using anti‑ISL1 and analyzed by immunoblotting using antibody against phosphorylated serine and ISL1 (HC: heavy chain, LC: light chain). b Alignments of the cluster of ISL1; S269 fit the CDK1 phosphorylation consensus, and (S/T)PX(R/K) sequences (red shade) are shown

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: CDK1 mediates ISL1 phosphorylation at serine 269. a MGC803 cells transfected with plasmid encoding CDK1 or control vector (left), negative control or CDK1 si-RNA (right). Lysate was analyzed by immunoblotting with antibodies against the indicated proteins. b MGC803 cells were transfected with plasmids encoding dominant‑negative mutant CDK1 (DN‑CDK1) or CDK1, and lysate was analyzed by immunoblotting with antibodies against the indicated proteins. c MKN28 cells expressing wild-type ISL1 or ISL1-S269A were transfected with plasmid encoding CDK1 or control vector. Cell lysates were immunoblotted with antibodies against the indicated proteins. d MKN28 cells were transfected with plasmids encoding wild-type ISL1 or ISL1-S269A and treated with CDK1 inhibitor RO-3306 or DMSO. Lysates were analyzed by immunoblotting with antibodies against the indicated proteins. GAPDH served as the internal control. e Representative IHC staining of ISL1-S269-p expression in GC (right, n = 60) and paired adjacent tissues (left, n = 60). f Relationship between expression of ISL1-S629-p and overall survival in gastric cancer

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Plasmid Preparation, Negative Control, Western Blot, Dominant Negative Mutation, Expressing, Immunohistochemistry

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: Phosphorylated ISL1 showed improved localization on cyclinB1 and cyclinB2 promoters. a ChIP assay analysis and PCR gel electrophoresis analysis of ISL-1 recruitment to the cyclinB1 and cyclinB2 promoters in MGC803 cells. b ChIP-re-IP assay was performed with anti-ISL-1 or rabbit IgG antibodies and then with anti-CDK1 or IgG antibodies for immunoprecipitation using chromatin harvested from MGC803 cells. c , d Quantitative chromatin immunoprecipitation analysis of cyclinB1 and cyclinB2 promoters in MKN28 cell line transfected with control vector or expressing wild-type ISL1 or ISL1-S269A plasmids. Data are expressed as mean ± s. d. from three individual experiments. (* p < 0.05, WT vs. control, # p < 0.05, S269A vs. control, $ p < 0.05, WT vs. S269A)

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Nucleic Acid Electrophoresis, Immunoprecipitation, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Expressing

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: Phosphorylation Ser 269 of ISL1 increases transactivation potency. a , b Effect of CDK1 activity on the transcriptional activity of wild-type ISL1 or S269A ISL1. Transcriptional activity of wild-type ISL1 and S269A ISL1 on cyclinB1-luc and cyclinB2-luc determined by luciferase reporter assay in MKN28 cells. c Cell proliferation was studied by CCK-8 analysis of MKN28 cells transfected with plasmids encoding control vector and wild-type ISL1 or ISL1-S269A. d Cell cycle distributions were analyzed by flow cytometry in MKN28 transfected with plasmids encoding control vector and wild-type ISL1 or ISL1-S269A. The data represent 3 independent experiments, each performed in triplicate. Each bar represents mean ± SD. (* p < 0.05, WT vs. control, # p < 0.05, S269A vs. control, $ p < 0.05, WT vs. S269A)

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Luciferase, Reporter Assay, CCK-8 Assay, Transfection, Plasmid Preparation, Flow Cytometry

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: Phosphorylation of ISL1 increases self-stability but does not increase its interaction with CDK1. a NIH3T3 cells were transfected with wild-type ISL1 or ISL1-S269A plasmids. After 48 h, cells were treated with CHX (100 µg/ml) for 12 h. Whole cell extracts were harvested for western blotting. b Co-IP assays were performed in NIH3T3 cells to detect the interaction between ISL1 (wild-type or mutants) and CDK1. GAPDH served as a loading and negative control. c Schematic representation of positive feedback regulation by the ISL1-CDK1/cyclin B1/2 complex in GC. Full arrow: confirmed; broken arrow: to be verified

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Western Blot, Co-Immunoprecipitation Assay, Negative Control

Journal: Developmental cell

Article Title: Low level saturated fatty acid palmitate benefits liver cells by boosting mitochondrial metabolism via CDK1-SIRT3-CPT2 cascade

doi: 10.1016/j.devcel.2019.11.012

Figure Lengend Snippet: (A) Total and mitochondrial cyclin B1 and activated CDK1 (pCDK1-T161) in AML12 cells treated with LPA for 24 and 48 h.

Article Snippet: Rabbit polyclonal anti-Phospho-CDK1 (Thr161) , Abcam , Cat# ab47329; RRID: AB_2260040.

Techniques:

Journal: Developmental cell

Article Title: Low level saturated fatty acid palmitate benefits liver cells by boosting mitochondrial metabolism via CDK1-SIRT3-CPT2 cascade

doi: 10.1016/j.devcel.2019.11.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Phospho-CDK1 (Thr161) , Abcam , Cat# ab47329; RRID: AB_2260040.

Techniques: Recombinant, Lysis, Luciferase, Protease Inhibitor, Magnetic Beads, Plasmid Preparation, Avidin-Biotin Assay, Blocking Assay, Mutagenesis, AST Assay, Software, Immunofluorescence, CRISPR

Journal: Developmental cell

Article Title: Low level saturated fatty acid palmitate benefits liver cells by boosting mitochondrial metabolism via CDK1-SIRT3-CPT2 cascade

doi: 10.1016/j.devcel.2019.11.012

Figure Lengend Snippet: (A) Total and mitochondrial cyclin B1 and activated CDK1 (pCDK1-T161) in AML12 cells treated with LPA for 24 and 48 h.

Article Snippet: Rabbit polyclonal anti-Phospho-Cdc2 (Thr161) , Cell Signaling , Cat# 9114; RRID: AB_2260040.

Techniques:

Journal: Developmental cell

Article Title: Low level saturated fatty acid palmitate benefits liver cells by boosting mitochondrial metabolism via CDK1-SIRT3-CPT2 cascade

doi: 10.1016/j.devcel.2019.11.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Phospho-Cdc2 (Thr161) , Cell Signaling , Cat# 9114; RRID: AB_2260040.

Techniques: Recombinant, Lysis, Luciferase, Protease Inhibitor, Magnetic Beads, Plasmid Preparation, Avidin-Biotin Assay, Blocking Assay, Mutagenesis, AST Assay, Software, Immunofluorescence, CRISPR

Journal: Cell Death Discovery

Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

doi: 10.1038/s41420-018-0044-7

Figure Lengend Snippet: a Scheme of the experimental protocol. b TTC-stained rostral and caudal brain sections from vehicle-treated Ctrl and Cdk1-cKO mice and R-roscovitine-treated Ctrl mice after 1-h MCAO and 24 h of reperfusion. Scale bar = 1 mm. c Rostral and caudal quantification of infarct size (% brain area) (data represent means ± SEM; n = 6-10; *p<0.05; **p<0.005; *** p < 0.001; ANOVA with Bonferroni’s post hoc). d Quantification of neuroscore scale (data represents means ± SEM; n = 6–10; * p < 0.01; ANOVA with Bonferroni’s post hoc)

Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

Techniques: Staining

Journal: Cell Death Discovery

Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

doi: 10.1038/s41420-018-0044-7

Figure Lengend Snippet: a Scheme of the experimental protocol. b Representative confocal microscopy images and quantitative results showing no differences in the number of CC3-positive cells between normoxic and OGD condition 4 h following OGD, scale bar = 30 µm and field size = 120 µm × 70 µm (data represent means ± SEM; n = 4; ns; Student’s t -test). c Representative confocal microscopy images and quantitative results illustrating significant differences in the number of CC3-positive cells (data represent means ± SEM; n = 4; *** p < 0.001; Student’s t -test) and survival (MTT assay) (data represent means ± SEM; n = 5; * p < 0.01; Student’s t -test) between normoxic and OGD condition at 24 h following OGD. d Representative confocal images showing OGD-induced Cdk1 expression 4 h following OGD compared with normoxic condition, scale bar = 30 µm. Quantitative results represent the percentage of living (CC3-negative) neurons expressing Cdk1, field size = 120 µm × 70 µm (data represent means ± SEM; n = 5; *** p < 0.001; Student’s t -test). Western blot analysis demonstrated increased Cdk1 expression 4 h following OGD

Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

Techniques: Confocal Microscopy, MTT Assay, Expressing, Western Blot

Journal: Cell Death Discovery

Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

doi: 10.1038/s41420-018-0044-7

Figure Lengend Snippet: a Representative confocal images illustrating OGD-induced Cdk1 neuronal expression, scale bar = 30 µm. Quantitative results represent the percentage of Tuj1-positive living neurons (green) expressing Cdk1 (red), field size = 140 µm × 90 µm (data represent means ± SEM; n = 4; *** p < 0.001; ANOVA with Bonferroni’s post hoc). Western blot analysis indicated increased Cdk1 expression 1, 2 and 4 h following OGD. b Representative confocal images illustrating the number of Tuj1-positive (green) and CC3-positive (red) cells in the different conditions/genotypes 24 h following OGD, scale bar = 30 µm. Quantitative results for neuronal survival represents the number of Tuj1-positive/CC3-negative cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 5; *** p < 0.001; one-way ANOVA with Bonferroni’s post hoc). MTT assay confirm significant difference regarding neuronal survival between the different conditions 24 h following OGD (data represent means ± SEM; n = 5; *p<0.05; **p<0.01 ; one-way ANOVA with Bonferroni’s post hoc). Quantitative results for neuronal death represent the number of CC3-positive cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 4; *p<0.05; ** p < 0.005; one-way ANOVA). Western blot analysis showed decreased C-Parp (89 kDa) protein expression in Cdk1-cKO OGD-treated cultures as compared with Ctrl OGD-treated cultures

Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

Techniques: Expressing, Western Blot, MTT Assay

Journal: Cell Death Discovery

Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

doi: 10.1038/s41420-018-0044-7

Figure Lengend Snippet: a Photography of 2 mm thick TTC-stained mouse brain coronal section 24 h after a transient 1 h MCAO, showing the healthy cortex (red), ischemic core (white) and peri-infarct area (between red and white) in the ipsilateral cortex. b Immunohistochemistry images showing NeuN staining in the healthy part of the ipsilateral cortex and the absence of NeuN staining in the ischemic core. In between, the peri-infarct area shows re-expression of Cdk1 and P-Cdk1. Scale bar = 100 µm

Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

Techniques: Staining, Immunohistochemistry, Expressing