Journal: Science Advances

Article Title: Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement

doi: 10.1126/sciadv.abq6480

Figure Lengend Snippet: ( A ) Percentage of WT leader cells that dissociate in confining microchannels under taxol ( n ≥ 369 cells, four experiments) or colchicine ( n ≥ 514 cells, five experiments) treatment. Data represent means ± SD. ( B and C ) Representative confocal images (B) and directionality (C) of GFP-tubulin in cells on 2D or in microchannels. 0° is parallel to the direction of migration. Data represent means ± SD for n ≥ 32 cells from three experiments. ( D ) Representative confocal images of cells expressing EB1-GFP on 2D (left) or in microchannels (right). ( E ) Microtubule polymerization rates of EB1-GFP in cells on 2D and in microchannels. Data represent means ± SD for n = 30 cells from three experiments. ( F ) Donor fluorescence lifetime of RhoA activity following colchicine treatment of cells on 2D and in confinement. Data represent means ± SD for n ≥ 18 cells from three experiments. ( G ) Representative confocal images of cells expressing GFP-GEF-H1 on 2D (left) or in microchannels (right). ( H ) Mean intensity of GFP-GEF-H1 at the front and rear of cells on 2D, upon cell entry into confinement, and at the time of dissociation. Values were normalized to the mean intensity of the cytoplasm in each cell. Data represent means ± SD for n = 13 to 39 cells from five experiments. ( I ) Donor fluorescence lifetime of RhoA activity in SC or GEF-H1 shRNA cells on 2D and in confinement. Data represent means ± SD for n ≥ 43 cells from four experiments. ( J ) Percentage of SC or GEF-H1 shRNA leader cells that dissociate in confining microchannels. Data represent means ± SD for n ≥ 272 leader cells from three experiments. Tests performed: Student’s t test (A, E, H, and J) (during entry), Student’s t test after log transformation (H) (during dissociation), one-way ANOVA test (F and I), or two-way ANOVA (C) (see table S3). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Scale bars, 6 μm. Cell model: A431.

Article Snippet: Lentiviral shRNAs targeting integrin β1, E-cadherin (CDH1), and GEF-H1 (ARHGEF2) were generated by subcloning the following sequences into pLKO.1 puro (plasmid #8453, Addgene): ITGβ1_sense, 5′-CCG GGT TGT AGG AAG AGG GAT AAT ACT CGA GTA TTA TCC CTC TTC CTA CAA ATT TTT G-3′; ITGβ1_anti-sense, 5′-AAT TCA AAA ATT TGT AGG AAG AGG GAT AAT ACT CGA GTA TTA TCC CTC TTC CTA CAA C-3′; CDH1_sense, 5′-CCG GGC ACC CGG GAC AAC GTT TAT TCT CGA GAA TAA ACG TTG TCC CGG GTG TTT TTT G-3′; CDH1_anti-sense, 5′-AAT TCA AAA AAC ACC CGG GAC AAC GTT TAT TCT CGA GAA TAA ACG TTG TCC CGG GTG C-3′; ARHGEF2_sense, 5′-CCG GCG CTC TGT CCA TCG AAA CTT TCT CGA GAA AGT TTC GAT GGA CAG AGC GTT TTT G-3′; and ARHGEF2_anti-sense, 5′-AAT TCA AAA ACG CTC TGT CCA TCG AAA CTT TCT CGA GAA AGT TTC GAT GGA CAG AGC G-3′.

Techniques: Migration, Expressing, Fluorescence, Activity Assay, shRNA, Transformation Assay

Journal: Science Advances

Article Title: Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement

doi: 10.1126/sciadv.abq6480

Figure Lengend Snippet: Confinement induces slow microtubule polymerization, which increases the cytosolic fraction of unbound GEF-H1. Concurrently, Ect2 and RacGAP1 exit the nucleus. Together, these three proteins activate RhoA, which relocalizes to cell-cell junctions. Actomyosin contraction via ROCK and MIIA promotes junctional collapse, E-cadherin disengagement, and leader cell detachment.

Article Snippet: Lentiviral shRNAs targeting integrin β1, E-cadherin (CDH1), and GEF-H1 (ARHGEF2) were generated by subcloning the following sequences into pLKO.1 puro (plasmid #8453, Addgene): ITGβ1_sense, 5′-CCG GGT TGT AGG AAG AGG GAT AAT ACT CGA GTA TTA TCC CTC TTC CTA CAA ATT TTT G-3′; ITGβ1_anti-sense, 5′-AAT TCA AAA ATT TGT AGG AAG AGG GAT AAT ACT CGA GTA TTA TCC CTC TTC CTA CAA C-3′; CDH1_sense, 5′-CCG GGC ACC CGG GAC AAC GTT TAT TCT CGA GAA TAA ACG TTG TCC CGG GTG TTT TTT G-3′; CDH1_anti-sense, 5′-AAT TCA AAA AAC ACC CGG GAC AAC GTT TAT TCT CGA GAA TAA ACG TTG TCC CGG GTG C-3′; ARHGEF2_sense, 5′-CCG GCG CTC TGT CCA TCG AAA CTT TCT CGA GAA AGT TTC GAT GGA CAG AGC GTT TTT G-3′; and ARHGEF2_anti-sense, 5′-AAT TCA AAA ACG CTC TGT CCA TCG AAA CTT TCT CGA GAA AGT TTC GAT GGA CAG AGC G-3′.

Techniques:

Journal: Cell

Article Title: Neuronal Inactivity Co-opts LTP Machinery to Drive Potassium Channel Splicing and Homeostatic Spike Widening

doi: 10.1016/j.cell.2020.05.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: mouse βCaMKK construct: Lentiviral CaMKKbeta , Green et al., 2011b , Addgene Plasmid #33322; RRID:Addgene_33322.

Techniques: Recombinant, Mutagenesis, Immunoprecipitation, Isolation, Protein Extraction, Lysis, Labeling, Construct, Plasmid Preparation, shRNA, Software

Journal: iScience

Article Title: CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

doi: 10.1016/j.isci.2025.114255

Figure Lengend Snippet: CaMKKβ regulates genes critical for endothelial integrity via DNA methylation (A) Defective expression of VE-cadherin and Tie2 in Camkkβ −/− mice. Total lung lysates from WT, Camkkβ +/− , and Camkkβ −/− mice were used for IB. Representative blot is shown; N = 3 mice/genotype. (B) CaMKKβ regulates the transcription of genes responsible for endothelial barrier integrity. Lung tissue (LT) mRNA levels were determined by qRT-PCR. N ≥ 3 mice per group; ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, WT vs. Camkkβ −/− (unpaired t test). (C) Expression of WT-CaMKKβ in lung endothelial cells of Camkkβ −/− mice rescued the expression of VE-cadherin and Tie2. Camkkβ −/− mice were injected with liposome-CaMKKβ plasmid (1 μg plasmid/gm body weight) complexes containing either WT-CaMKKβ or the kinase defective CaMKKβ ( K193A -CaMKKβ) mutant. At 96 h after injection, the lungs harvested were used for IB analysis. N = 3 mice per group; representative blot is shown. ∗ p < 0.05, ∗∗ p < 0.01, WT vs. WT-CaMKKβ or K193A-CaMKKβ (two-way ANOVA). (D) DNA methyltransferase inhibition restored the expression of VE-cadherin and Tie2 in Camkkβ −/− mice. WT and Camkkβ −/− mice were injected with 5-AZA (1 mg/kg; i.p.) or vehicle for 5 consecutive days and the lungs were harvested used for IB analysis. N = 3 mice per group; representative blot is shown; n.s, not significant; ∗∗∗∗ p < 0.0001. ∗ p < 0.05, ∗∗ p < 0.01, WT vs. WT-CaMKKβ or K193A -CaMKKβ (two-way ANOVA). (E) WT and Camkkβ −/− mice were injected with 5-AZA as above were treated with LPS (5 mg/kg, i.p.) for 0 and 6 h and lung vascular permeability was determined via measuring EBA uptake. N = 4 mice/group; n.s, not significant, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, WT vs. Camkkβ −/− (two-way ANOVA).

Article Snippet: CaMKKβ K193A mutant plasmid , Addgene , Cat# 33323.

Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Injection, Plasmid Preparation, Mutagenesis, Inhibition, Permeability

Journal: iScience

Article Title: CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

doi: 10.1016/j.isci.2025.114255

Figure Lengend Snippet: CaMKKβ deficiency leads to the hyper-methylation of the Elf2 transcription factor gene (A) DNA sequence reveals the low levels of CpG sites in the promoter regions of mouse Tie2 and VE-cadherin (Cdh5). (B) Volcano plot shows changes in the methylation level of genes in lungs of Camkkβ +/+ and CamKKβ −/− mice. The ratio of CpG methylated sites in genes (+/− 2 kb of the TSS) of Camkkβ +/+ /Camkkβ −/− mice were presented (fold changes vs. p -value). (C) Quantified data show hyper-methylation of Elf2 gene promoter in CamKKβ −/− mice. N = 3 samples per genotype; ∗ p < 0.05 (unpaired t test). (D) DNA sequence analysis reveals the abundance of CpG islands and CpG sites in the promoter regions of human and mouse Elf2 genes. (E) WT and Camkkβ −/− mice lung mRNA levels of Elf2 and Elf1 were determined by qRT-PCR. N = 3 mice per genotype; ns, not significant, ∗∗∗ p < 0.001 (unpaired t test). (F) DNA methyl transferase inhibition restores Elf2 expression in Camkkβ −/− mice. WT and Camkkβ −/− mice were injected with 5-AZA (1 mg/kg, i.p.) or vehicle for 5 days as described above in C. Lungs were harvested and used for IB. N = 3 mice per group; representative blot is shown. (G) Expression of WT-CaMKKβ in EC of Camkkβ −/− mice rescued Elf2 expression. Camkkβ −/− mice were injected with liposome-CaMKKβ plasmid (1 μg plasmid/g body weight) complexes containing either WT-CaMKKβ or the kinase defective CaMKKβ ( K193A -CaMKKβ) mutant. At 96 h after injection, lungs were harvested and used for IB analysis. N = 3 mice per group; representative blot is shown.

Article Snippet: CaMKKβ K193A mutant plasmid , Addgene , Cat# 33323.

Techniques: Methylation, Sequencing, Quantitative RT-PCR, Inhibition, Expressing, Injection, Plasmid Preparation, Mutagenesis

Journal: Molecular Cell

Article Title: Genomic RNA Elements Drive Phase Separation of the SARS-CoV-2 Nucleocapsid

doi: 10.1016/j.molcel.2020.11.041

Figure Lengend Snippet:

Article Snippet: 25ug of plasmid DNA for each Nucleocapsid GFP Spark (Sino biological VG40588-ACGLN) and H2BmCherry (from Jun Lu lab Yale University) was co-transfected using calcium phosphate.

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Software, Sequencing