Journal: EMBO Reports

Article Title: The L27 domain of MPP7 enhances TAZ-YY1 cooperation to renew muscle stem cells

doi: 10.1038/s44319-024-00305-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Jasplakinolide , Tocris Bioscience , Catalog #2792.

Techniques: Recombinant, Modification, Magnetic Beads, Sequencing, Blocking Assay, Plasmid Preparation, Electron Microscopy, Software, Reporter Assay, Imaging, In Situ

Journal: Research

Article Title: The Mechanics of Tumor Cells Dictate Malignancy via Cytoskeleton-Mediated APC/Wnt/β-Catenin Signaling

doi: 10.34133/research.0224

Figure Lengend Snippet: Softening cells promotes self-renewal and tumorigenicity. (A) Phosphorylation of MLC and cell stiffness after knocking down MLCK and mDia1 using 10 nM siRNAs. n > 80 cells. (B and C) Formation of tumor spheroids in sphere formation assay (SFA) and soft agar assay after HCC cells were softened. n = 3 and 20 for soft agar assay and SFA, respectively. (D) Growth of tumor spheroids generated by softened tumor cells in soft fibrin. Representative of 3 independent experiments. (E) Fractions of EpCAM + , CD90 + , and CD133 + HCC cells. n = 4, 5, and 3 for EpCAM, CD90, and CD133, respectively. (F and G) Colony formation and growth in soft agar and soft fibrin after Huh-7 cells were transfected with the siRNAs of MLCK and mDia1 and then treated with 20 nM jasplakinolide (Jas) or 1 nM narciclasine (Narci). n = 3. (H and I) Tumor growth after the softened Huh-7 cells were inoculated subcutaneously (S.C.) into the flank of nude mice. n = 5 for each condition. After the experiment, the tumor tissues were retrieved and weighted. Scale bar, 1 cm. (J) Tumor growth after the softened MHCC97L cells were prelabeled with luciferase and then inoculated into the liver of nude mice. n = 13 for each condition. (K) Tumor incidence of the softened tumor cells in the limiting dilution assay. In total, 100, 1,000, 10,000, and 100,000 of control and softened Huh-7 cells were inoculated subcutaneously into the flank of nude mice. The tumor incidence was measured at week 8. The stem cell frequency and P value (chi-square test) were calculated using the tools at http://bioinf.wehi.edu.au/software/elda/ . Mean ± SEM. Analysis of variance (ANOVA) was used for the statistical analysis together with the post hoc Bonferroni test. * P < 0.05; ** P < 0.01; *** P < 0.001; # P < 0.05; ## P < 0.01; ### P < 0.001. * or #, the comparison between “si-MLCK” or “si-mDia1” and “Control”. ns: no significance.

Article Snippet: To soften tumor cells, HCC cells were pretreated with different doses of Y-27632 (2 and 20 μM; Selleckchem), blebbistatin (6 and 50 μM; Selleckchem), and cytochalasin D (0.1 and 1 μM; Selleckchem) for 48 h. To stiffen them, the cells were pretreated with 20 nM jasplakinolide (Tocris, #2792/100U) or 1 nM narciclasine (MedChemExpress) for 12 h. Nocodazole (1 and 6 μM; Abcam) and withaferin A (WFA; Abcam; 1, 2, and 5 μM) were used to disrupt microtubules and vimentin, respectively.

Techniques: Tube Formation Assay, Soft Agar Assay, Generated, Transfection, Luciferase, Limiting Dilution Assay, Control, Software, Comparison

Journal: eLife

Article Title: Combinatorial deployment of F-actin regulators to build complex 3D actin structures in vivo

doi: 10.7554/eLife.63046

Figure Lengend Snippet: ( A, B ) FRAP T50 and immobile fraction of F-actin in control and indicated actin regulator compromised embryos in cycle 11. ns: not significant, *: p<0.05, **: p<0.005, ***: p<0.0005. Control: n = 13; ArpC4 shRNA: n = 14; Dia shRNA: n = 9; DPod1 shRNA: n = 9; Cortactin shRNA: n = 8; Coronin shRNA: n = 8; Scar shRNA: n = 7 ( C, D ) FRAP T50 and immobile fraction of F-actin in control (n = 13), Latrunculin B (LatB) (n = 9), and Jasplakinolide (Jasp) injected embryos (n = 8), or performed in ArpC4 shRNA embryos (n = 9) and ArpC4 shRNA with Jasp injection (n = 8). ( E ) Individual FRAP representative examples in the indicated backgrounds. *: p<0.05, ***: p<0.0005.

Article Snippet: Jasplakinolide , Santa Cruz Biotech , Cat# sc202191.

Techniques: Control, shRNA, Injection

Journal: eLife

Article Title: Combinatorial deployment of F-actin regulators to build complex 3D actin structures in vivo

doi: 10.7554/eLife.63046

Figure Lengend Snippet: Reagents.

Article Snippet: Jasplakinolide , Santa Cruz Biotech , Cat# sc202191.

Techniques: Electron Microscopy, Reverse Transcription, SYBR Green Assay, Mutagenesis, Software, CRISPR, Construct

Journal: bioRxiv

Article Title: Morphological profiling of human T and NK lymphocytes identifies actin-mediated control of the immunological synapse

doi: 10.1101/2020.01.17.910091

Figure Lengend Snippet: Graphs representing the fold change of IS parameters and representative images of NK-92 cells seeded on ICAM-1, anti-NKp30 and anti-NKp46, stained for F-actin (green), perforin granules (yellow) and nuclei (DAPI) and treated with three concentrations of a . Latrunculin b. Jasplakinolide, c. Blebbistatin, d. Y-27632, e. CK-968, f. Wiskostatin, and g. SMIFH2 with respect to the untreated control. h. Untreated control. The data represent the mean of triplicates for each concentration (1425-5541 cells). Scale bars: 10 µm.

Article Snippet: NK-92 were treated with 5, 10 and 50 µM Blebbistatin, 10, 25 and 50 µM CK-869 (Merck), 0.1, 1 and 2.5 µM Jasplakinolide (Merck), 0.1, 0.25 and 0.5 µM Latrunculin B (Merck), 50, 100 and 250 µM SMIFH2 (Merck), 10 50 and 100 µM Wiskostatin (Merck) and 5, 10 and 25 µM Y-27632 (Merck) for 30 min at 37°C and washed twice in PBS before seeding onto the plates and letting them adhere for 30 min.

Techniques: Staining, Concentration Assay

Journal: bioRxiv

Article Title: Morphological profiling of human T and NK lymphocytes identifies actin-mediated control of the immunological synapse

doi: 10.1101/2020.01.17.910091

Figure Lengend Snippet: Drug-treated NK-92 cell images were analysed with CellProfiler for an array of measurements and visualized using UMAP to position drug treated cells with respect to untreated cells from the same row. a . Latrunculin b. Jasplakinolide, c. Blebbistatin, d. Y-27632, e. CK-968, f. Wiskostatin, and g. SMIFH2. h. Confusion matrix and class-wise performance on held-out data of a random forest model trained to predict drug treatment based on the morphology of NK-92 cells seeded on ICAM-1, anti-NKp30 and anti-NKp46. i-j Total and average importance for the prediction of morphological features per measurement type and biological object described of NK-92 cells seeded on ICAM-1, anti-NKp30 and anti-NKp46 and treated with i. CK-869 or j. SMIFH2.

Article Snippet: NK-92 were treated with 5, 10 and 50 µM Blebbistatin, 10, 25 and 50 µM CK-869 (Merck), 0.1, 1 and 2.5 µM Jasplakinolide (Merck), 0.1, 0.25 and 0.5 µM Latrunculin B (Merck), 50, 100 and 250 µM SMIFH2 (Merck), 10 50 and 100 µM Wiskostatin (Merck) and 5, 10 and 25 µM Y-27632 (Merck) for 30 min at 37°C and washed twice in PBS before seeding onto the plates and letting them adhere for 30 min.

Techniques:

Journal: bioRxiv

Article Title: Single-Molecule Imaging and Microfluidic Platform Reveal Molecular Mechanisms of Leukemic Cell Rolling

doi: 10.1101/2020.04.29.069617

Figure Lengend Snippet: Detachment of cell membrane from actin cytoskeleton during the tether formation. (a) Time-lapse fluorescence images of the PSGL-1 molecule (immunostained by Alexa-Fluor-555-conjugated anti-PSGL-1 antibody) on the KG1a cell captured during cell rolling over E-selectin, which show the merger of the multiple tethers. The white arrowheads show the regions in which the merger of the tethers occurs. (b) Two-color super-resolution fluorescence localization microscopy image of CD44 (cyan, immunostained by anti-CD44 515 antibody followed by Alexa-Fluor-647-conjugated secondary antibodies) and actin (yellow, labeled by Alexa-Fluor-488-conjugated phalloidin) on the control KG1a cell. The insets show enlarged views of the yellow regions. (c) Two-color fluorescence images of the cell membrane (cyan, stained by Vybrant DiO dye) and actin (yellow, labeled by silicon-rhodamine-conjugated jasplakinolide) on the slings of the KG1a cells formed during cell rolling over E-selectin. The white arrowheads show the regions in which the patches of actin colocalize with the slings. All the fluorescence images of the rolling cells were captured by injecting KG1a cells into the microfluidic chambers at a shear stress of 2 dyne cm -2 (0.2 Pa) whose surface was coated by the rh E-selectin at a density of 15 molecules μm -2 .

Article Snippet: For the two-color fluorescence imaging of the cell membrane and the selectin ligands (either CD44 or PSGL-1), the Vybrant DiO-stained KG1a cells were incubated with the Alexa-Fluor-647-conjugated anti-CD44 or anti-PSGL-1 antibodies (5 μg for each 10 6 cells) on ice for 45 min. We used silicon rhodamine (SiR)-conjugated jasplakinolide (Cytoskeleton, Inc.) for staining actin cytoskeleton of the live KG1a cells.

Techniques: Fluorescence, Microscopy, Labeling, Staining