Journal: PLoS ONE

Article Title: Functions of Nonmuscle Myosin II in Assembly of the Cellular Contractile System

doi: 10.1371/journal.pone.0040814

Figure Lengend Snippet: (A,B) Cell surface topography revealed by platinum replica EM of non-extracted untreated (A) or 100 µM blebbistatin (BS)-treated cells (B). (C,D) Fluorescence microscopy of phalloidin-stained F-actin and immunostained α-actinin in detergent-extracted untreated (C) or 100 µM blebbistatin-treated cells (D). (E) Fractions of the cell perimeter occupied by lamellipodia and ruffles in untreated cells, cells treated with 75 µM or 100 µM blebbistatin, and cells recovering from treatment with 100 µM blebbistatin for indicated periods of time in minutes. Error bars, SD. (F,G) Fluorescence microscopy of phalloidin-stained F-actin and immunostained vinculin in detergent-extracted untreated (F) or 100 µM blebbistatin-treated (G) cells. Boxed regions in C, D, E, and G are zoomed in bottom panels. Scale bars, 2 µm (A,B) and 20 µm (C,D,F,G).

Article Snippet: The following primary antibodies were used: mouse monoclonal α-actinin (Cytoskeleton), mouse monoclonal vinculin (Sigma), rabbit polyclonal NMII from bovine spleen , rabbit polyclonal mono- (Ser19) or double- (Thr18/Ser19) phosphorylated MRLC (Cell Signaling Technology, a gift from Dr. C. Chen, University of Pennsylvania).

Techniques: Fluorescence, Microscopy, Staining

Journal: PLoS ONE

Article Title: Functions of Nonmuscle Myosin II in Assembly of the Cellular Contractile System

doi: 10.1371/journal.pone.0040814

Figure Lengend Snippet: (1) NMII activation : Inactive NMII molecules diffuse to lamellipodia, where they are activated by double phosphorylation of MRLC. (2) Focal complex formation: Active unpolymerized NMII molecules bind actin filaments in lamellipodia and undergo the retrograde flow with them. If two NMII heads bind different actin filaments, one of which is anchored to a nascent adhesion, the resulting strain stabilizes the nascent adhesion and promotes its transformation to a focal complex. Long filaments in filopodia can encounter more NMII molecules increasing a probability of focal complex formation under filopodial bundles. (3) Assembly of NMII filaments: NMII molecules pulling on actin filaments attached to focal complexes experience a greater load, which triggers tension-dependent NMII polymerization at these sites. (4) Stress fiber formation: Multivalent NMII filaments exert large forces sufficient to promote maturation of focal complexes to focal adhesions. They also cross-link and align disordered actin filaments into bundles producing stress fibers. However, additional events, such as recruitment of α-actinin, are needed for the formation of semi-sarcomeric pattern in stress fibers.

Article Snippet: The following primary antibodies were used: mouse monoclonal α-actinin (Cytoskeleton), mouse monoclonal vinculin (Sigma), rabbit polyclonal NMII from bovine spleen , rabbit polyclonal mono- (Ser19) or double- (Thr18/Ser19) phosphorylated MRLC (Cell Signaling Technology, a gift from Dr. C. Chen, University of Pennsylvania).

Techniques: Activation Assay, Transformation Assay

Journal: Molecules

Article Title: Impaired Expression of Cytoplasmic Actins Leads to Chromosomal Instability of MDA-MB-231 Basal-Like Mammary Gland Cancer Cell Line

doi: 10.3390/molecules26082151

Figure Lengend Snippet: Figure 1. Downregulation of β- or γ-actin expression by corresponding shRNAs in MDA-MB-231 breast cancer cells leads to phenotype changes. (A). Immunofluorescent staining of MDA-MB-231 cells with β- or γ-actin downregulation by corresponding shRNAs. Arrowheads mark leading edge with actin enrichment. Bar, 10 µm. (B). Downregulation of cytoplasmic β- or γ-actin in MDA-MB-231 cells. WB analysis. Graphs represent relative actins expression (Mean ± SEM). For cells with shRNA compared with control (β-actin): p = 0.0015 (shRNA to β-actin); p = 0.007 (shRNA to γ-actin); (γ-actin): p = 0.0037 (shRNA to β-actin); p = 0.0048 (shRNA to γ-actin); Mann–Whitney U test, n = 3. Values of p < 0.01 (**) were considered as statistically significant.

Article Snippet: Mouse monoclonal antibodies to: β-actin (MCA5775GA, AbD Serotec); γ-actin (MCA5776GA, AbD Serotec, Raleigh, NC, USA); pan actin (4968, Cell Signaling) were used.

Techniques: Expressing, Staining, shRNA, Control, MANN-WHITNEY

Journal: Molecules

Article Title: Impaired Expression of Cytoplasmic Actins Leads to Chromosomal Instability of MDA-MB-231 Basal-Like Mammary Gland Cancer Cell Line

doi: 10.3390/molecules26082151

Figure Lengend Snippet: Figure 2. Downregulation of β- or γ-actin expression by corresponding shRNAs in MDA-MB-231 breast cancer cells leads to cell cycle changes. (A). After 6d of β- or γ-actin depletion by corresponding shRNAs MDA-MB-231 cells were stained with propidium iodide. High content analysis of cell cycle distributions of flow cytometric data was performed after 6d of β- or γ-actin depletion by corresponding shRNAs in MDA-MB-231 cells. (B). The effects of β- or γ-actin depletion on quantitative ratio of cells in interphase and different mitotic phases were analyzed by IF cytometry. The Y-axis indicates the number of cells in specific phases. Student’s t-test, n = 3. Values of p < 0.01 (**), and p < 0.05 (*) were considered as statistically significant. (C). The effects of β- or γ-actin depletion on mitotic phases were analyzed by IF cytometry. The pie chart shows the proportion of cells in different phases of mitosis.

Article Snippet: Mouse monoclonal antibodies to: β-actin (MCA5775GA, AbD Serotec); γ-actin (MCA5776GA, AbD Serotec, Raleigh, NC, USA); pan actin (4968, Cell Signaling) were used.

Techniques: Expressing, Staining, High Content Screening, Cytometry

Journal: Molecules

Article Title: Impaired Expression of Cytoplasmic Actins Leads to Chromosomal Instability of MDA-MB-231 Basal-Like Mammary Gland Cancer Cell Line

doi: 10.3390/molecules26082151

Figure Lengend Snippet: Figure 3. Karyotypic analysis of MDA-MB-231 breast cancer cells. (A). A representative G-banded karyotype of MDA- MB-231 cell line. (B). Composite G-banded karyotype of the near-triploid cell line MDA-MB-231, showing structural and numerical changes. Arrows point to main chromosomal alterations. Mar—Marker chromosome. (C,D,F,G). The representa- tive chromosome aberrations in β-actin (C,D) and γ-actin (F,G) -depleted MDA-MB-231 cells: dicentric chromosomes (C,F) and acentric fragment (C), ring chromosomes (D,G). Arrows point to aberrations. E, H. Endoreduplication in β-actin (E) and γ-actin (H) -depleted MDA-MB-231 cell.

Article Snippet: Mouse monoclonal antibodies to: β-actin (MCA5775GA, AbD Serotec); γ-actin (MCA5776GA, AbD Serotec, Raleigh, NC, USA); pan actin (4968, Cell Signaling) were used.

Techniques: Marker