Journal: The Journal of Cell Biology

Article Title: Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO

doi: 10.1083/jcb.201605104

Figure Lengend Snippet: ARF1-GTP levels and podosome formation. (A) Quantification of ARF1-GTP levels by G-LISA assay in control, stimulated, and inhibitor-treated THP1 cells. Both TGFβ1 and PMA increased the fraction of GTP-bound ARF1 compared with control, whereas treatment with SecinH3 or BFA dramatically reduced it. Pooled results of three independent experiments are shown. Mean ± SD is indicated. (B) Disruption of podosomes labeled with mCherry-Utrophin (UtrCH) upon treatment with SecinH3 (top). Note that integrity of the Golgi apparatus labeled with GFP-mannosidase II was preserved in the same SecinH3-treated cell (bottom). See also Video 3. (C and D) Disruption of podosomes labeled with mCherry-vinculin by SecinH3 (C, top) and BFA (D, top). Although the effect of SecinH3 in these cells was not accompanied by changes in localization of ARF1 to the Golgi and cytoplasmic puncta (C, bottom), BFA disrupted both Golgi and ARF1 puncta (D, bottom); Bars, 5 µm. Insets (1 × 1 µm 2 ) show evolution of individual ARF1 puncta in each case. (E and F) Quantification of the effect of SecinH3 and BFA on number of podosomes per cell (E) and percentage of cells with more than 10 podosomes (F). (G) ARF1-GTP level increase in fibroblasts plated on a RGD-functionalized fluid lipid bilayer compared with fibroblasts plated on glass coverslip. Mean ± SD is indicated. (H) Effect of SecinH3 on the integrity of podosome-like structures formed by fibroblasts plated on fluid lipid bilayer. (I) Quantification of the disruptive effect of SecinH3 and BFA on podosome-like structures formed by fibroblasts on lipid bilayer. The percentage of podosome-forming cells significantly decreased upon treatment by each of the inhibitors. The data in E, F, and I are presented as indicated in the legend to . Pooled data of three independent experiments are presented for each group. The significance of the difference between groups was estimated by two-tailed Student’s t test. ns, P > 0.05 (nonsignificant); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: For drug inhibition studies, cells were treated with 30 μM SecinH3 (Tocris Bioscience), 10 μM Golgicide A (Santa Cruz Biotechnology, Inc.), 5 μg/ml BFA (Sigma-Aldrich), 30 μM Y-27632 (Sigma-Aldrich), 25 μM GM6001 (Enzo Life Sciences), and 2 μg/ml C3 transferase (Cytoskeleton, Inc.) in complete medium for 1–2 h or 4 h for GM6001 at 37°C with 5% CO 2 and subsequently fixed with 4% PFA.

Techniques: Control, Disruption, Labeling, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO

doi: 10.1083/jcb.201605104

Figure Lengend Snippet: Inhibition of ARF1 activity induces RhoA activation. (A–C) 1-h incubation of TGFβ1-stimulated THP1 cells with 30 µM SecinH3 led to increase in RhoA-GTP (A) but not Rac1-GTP (B) or Cdc42-GTP (C) fractions as indicated by Western blots after pull-down assay. (D–J) SIM visualization of podosome dynamics in TGFβ1-stimulated THP1. (D and E) Live imaging of cell stably transfected with GFP-MRLC to visualize myosin-II filaments and RFP-Lifeact to visualize podosome cores. (D) Cell treated with 30 µM SecinH3 shows an increase in myosin-IIA filament assembly (green) and disruption of podosomes (red). Enlarged image of white-boxed area (5 × 4.5 µm 2 ) of D shows colocalization between appearance of myosin-IIA filaments and podosome disruption (see Video 7). (E) Time course of podosome reappearance after addition of 30 µM ROCK inhibitor Y-27632 to cell incubated in SecinH3-containing medium. Note that podosomes (red) appeared after disassembly of myosin-II filaments (green). (F–J) TGFβ1-stimulated THP1 cells were transfected with either control scrambled siRNA (F and G) or with siRNA to NM-myosin-IIA heavy chain, MYH9 (I and J) and, after 48 h, treated with either 0.1% DMSO (F and I) or 30 µM SecinH3 (G and J) for 1 h. (F, G, I, and J) After fixation, the cells were stained with phalloidin (left) and antibodies to NM-myosin-IIA heavy chain (middle) and vinculin (right). Bars, 5 µm. (H) Western blot showing protein levels of NM-myosin-IIA heavy chain in control cells (transfected with scrambled siRNA) or in NM-myosin-IIA knockdown cells (transfected with MYH9 siRNA); α-tubulin was used as a loading control. (K and L) Effect of SecinH3 treatment of control and NM-myosin-IIA knockdown cells on number of podosomes per cell (K) and percentage of cells with more than 10 podosomes (L). The data in K and L are presented as indicated in the legend to . Pooled data of at least two independent experiments are presented for each group. The significance of the difference between groups was estimated by two-tailed Student’s t test. nonsignificant (ns), P > 0.05; ***, P ≤ 0.001.

Article Snippet: For drug inhibition studies, cells were treated with 30 μM SecinH3 (Tocris Bioscience), 10 μM Golgicide A (Santa Cruz Biotechnology, Inc.), 5 μg/ml BFA (Sigma-Aldrich), 30 μM Y-27632 (Sigma-Aldrich), 25 μM GM6001 (Enzo Life Sciences), and 2 μg/ml C3 transferase (Cytoskeleton, Inc.) in complete medium for 1–2 h or 4 h for GM6001 at 37°C with 5% CO 2 and subsequently fixed with 4% PFA.

Techniques: Inhibition, Activity Assay, Activation Assay, Incubation, Western Blot, Pull Down Assay, Imaging, Stable Transfection, Transfection, Disruption, Control, Staining, Knockdown, Two Tailed Test

Journal: Scientific Reports

Article Title: In PC3 prostate cancer cells ephrin receptors crosstalk to β 1 -integrins to strengthen adhesion to collagen type I

doi: 10.1038/srep08206

Figure Lengend Snippet: Graph depicts the adhesion force of PC3 cells after being in contact with collagen I for 60 s in the presence of inhibitors; wortmannin (100 nM, 0.01% final DMSO concentration), LY294002 (50 μM, 0.5%), NSC23766 (50 μM, 0.1%) and EHT1864 (100 μM, 0.13%), SecinH3 (20 μM, 0.1%), Akt inhibitor IV (1 μM, 0.1%), Akt inhibitor VIII (20 μM, 0.1%), GGTI2147 (10 μM, 1%), GGTI286 (10 μM, 1%), blebbistatin (10 μM, 0.02%), CTO4 (2 μg/mL, 1% glycerol), and Y27632 (10 μM, 0.1%). Blue and brown data points represent cells bound to ConA and ephrin-A1 coated cantilevers, respectively. Adhesion values recorded at contact times of 5 and 15 s are given in . Each dot represents the measurement of one PC3 cell. The number of cells assayed for each condition is given by . Bars mark mean force and standard deviation. Statistical differences to control experiments (untreated cells) were analyzed by Mann-Whitney U -tests ( P -values are given). Perturbations were considered significant if all inhibitors targeting the same protein significantly reduced the adhesion force.

Article Snippet: To inhibit cell signaling, wortmannin (100 nM; Sigma-Aldrich) , LY294002 (50 μM; Cell Signaling Technology) , NSC23766 (50 μM; Merck Millipore) , EHT 1864 (100 μM; R&D Systems) , GGTI 2147 or GGTI286 (10 μM; Merck Millipore) , Akt inhibitor IV (1 μM; Merck Millipore) , Akt inhibitor VIII (20 μM; Merck Millipore) , SecinH3 (20 μM; Merck Millipore) , blebbistatin (10 μM; Merck Millipore) or Y-27632 (10 μM; Merck Millipore) were added to the measurement media at the concentrations given.

Techniques: Concentration Assay, Standard Deviation, MANN-WHITNEY