arpc2  (Proteintech)

Journal: Biology Open

Article Title: Macrophages migrate persistently and directionally upon entering 2D confinement in the presence of extracellular matrix

doi: 10.1242/bio.061782

Figure Lengend Snippet: Arp2/3 complex disruption hinders haptotactic sensing but not confinement-induced directional migration. (A) Schematic depicting CK-666 experimental conditions. A 250 µg/ml FN gradient is generated as normal, but in this case the agarose is made up with 125 µM CK-666, and 60 µM CK-666 is included in center well, and both cell wells. Solid lines running through the schematic represent CK-666 polymerized into the gel. (B) Migration relative to the center well is represented by FMIx (left), velocity (middle), and persistence (right). Means and 95% confidence interval (for FMIx) or means and s.e.m. (velocity, persistence) are represented with black symbols, and all cell migration tracks are plotted and each experimental run is color- and shape-coded (circle, square, or triangle). Statistical analysis was done by Mann–Whitney test. *** P =0.0002 (FMIx), *** P =0.0010 (Persistence), ns=not significant. Close− n =250 tracks, Close+ n =194 tracks. Data were pooled from 3 independent experiments. (C) Schematic depicting experimental conditions for WT versus KO ( Arpc2−/− ) macrophages migrating concurrently on 250 µg/ml FN gradient. (D) Migration of WT and KO ( Arpc2−/− ) macrophages toward the center well is represented by FMIx (left), velocity (middle), and persistence (right). Means and 95% confidence interval (for FMIx) or means and s.e.m. (velocity, persistence) are represented with black symbols, and all cell migration tracks are plotted and each experimental run is color- and shape-coded (circle, square, or triangle). Statistical analysis was done by Kruskal–Wallis with Dunn multiple comparisons test. **** P <0.0001, *** P <0.0002, ns=not significant. WT− n =128, WT+ n =202, KO− n =95, KO+ n =97. These data were pooled from three separate experiments. (E) Representative images of differentially labeled WT (blue) versus KO (green) cell migrating against (close−) or along (close+) the fibronectin gradient. Representative KO ( Arpc2−/− ) cells migrating away from the gradient (close−) are marked with arrows colored light orange, light green, and dark green. KO ( Arpc2−/− ) cells migrating towards the gradient (close+) are marked with arrows colored red, orange, and yellow. Representative WT cells migrating away from the gradient (close−) are marked with arrows colored chestnut, brown, and light purple. WT cells migrating towards the gradient (close+) are marked with light blue, purple, and white. Scale bar:100 microns. The direction of the FN gradient is schematically depicted below.

Article Snippet: Arpc2 (Millipore Sigma, 07-227-I-25UG) and GAPDH (ThermoFisher Scientific, AM4300) antibodies were commercially sourced.

Techniques: Disruption, Migration, Generated, MANN-WHITNEY, Labeling

Journal: iScience

Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D

doi: 10.1016/j.isci.2025.112623

Figure Lengend Snippet: The Arp2/3-vinculin hybrid complex is required for pseudopod extension in 3D (A and B) Vinculin and kindlin-2, but not FAK knockout U2OS cells have spreading defects in 3D. Cells embedded in collagen gels were stained for F-actin, imaged and quantified as described in . PEI is normalized to wild-type control. Data are shown as mean ± s.d. Kruskal-Wallis test: ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001 (C) Direct Arp2/3-binding by vinculin is required for vinculin function during pseudopod extension in 3D. Vinculin KO cells were reconstituted with empty vector (ev), wild-type (wt), Arp3-binding mutant (Δ-Arp3) or Actin-binding mutants (Δ-IA or Δ-RE) in full-length vinculin. PEI values are normalized to the mean of the ev-distribution. Gray dotted line designates the spread levels of wild-type U2OS cells. Seven data points are outside the y axis limit, and the full graph is presented in E. Data are shown as mean ± s.d. Kruskal-Wallis test: ns = not significant, ∗ p ≤ 0.05, ∗∗∗ p ≤ 0.001. (D) Arp2/3-binding by vinculin is not essential for vinculin function during pseudopod extension in 2D. Vinculin KO cells were reconstituted with empty vector (ev), wild-type (wt) or indicated vinculin mutants (Arp3-binding mutant (Δ-Arp3), Actin-binding mutants (Δ-IA or Δ-RE)). PEI values are normalized to the mean of the ev-distribution. Gray dotted line designates the spread levels of wild-type U2OS cells. All vinculin constructs except the Δ-RE mutant were able to restore pseudopod formation of the vinculin KO cells comparable to that of wild-type U2OS cells. All data were collected from two to five independent experiments (N) and are shown as mean ± s.d. Each data point represents the median of the PEI of all cells within one image field of view (n) and normalized to the mean of the ev-distribution. Total number of quantified individual cells are indicated in brackets, but not used to compute any statistics. Kruskal-Wallis test: ns = not significant, ∗ p < 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. (E) Hybrid Arp2/3-vinculin complex formation is not abrogated in 2D. p34-ARC-vinculin PLA density was detected in cells seeded on collagen-coated glass coverslips, background subtracted (see ) and normalized to wild-type cells (wt). Each data point represents a cell. Data were collected from three independent experiments (N). Total number of quantified cells are indicated in brackets. Mean ± s.e.m. One-way ANOVA: ∗ p < 0.05, ∗∗∗∗ p ≤ 0.0001. (F) Formation of the hybrid Arp2/3-vinculin complex is sensitive to the extracellular environment. p34-ARC-vinculin PLA densities were analyzed as in (E) but from cells seeded on soft substrate (∼8 kPa). Each data point represents a cell. Data were collected from three independent experiments (N). Total number of quantified cells are indicated in brackets. Mean ± s.e.m. One-way ANOVA: ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Subsequently, the samples were stained with rabbit anti-p34-ARC/ARPC2 (Millipore; 07–227; 1:250) and mouse anti-vinculin (Sigma-Aldrich; V9264; 1:500) for 1 h at RT, followed by 1 h RT incubation with the Duolink PLA probes, 30 min ligation and 200 min amplification at 37°C in a humidified chamber.

Techniques: Knock-Out, Staining, Control, Binding Assay, Plasmid Preparation, Mutagenesis, Construct