Figure 3. ARF1-GTP levels and podosome... | Rockefeller University Press

Figure 3.

$Figure 3. 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 µm2) 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 Fig. 1. 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.$

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 µm²) 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 Fig. 1. 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.