Depletion of endogenous ARF1 disrupts podosomes. (A) Western blot showing ARF1 levels in cells treated with scrambled (control) or ARF1 siRNA; α-tubulin was used as a loading control. (B and C) ARF1 knockdown leads to disruption of podosomes but not the Golgi apparatus. Actin labeled with phalloidin (left) and vinculin visualized by antibody staining (right) in control (B) and ARF1 siRNA-transfected (C) THP1 cells 48 h after TGFβ1 stimulation. The Golgi apparatus in the same cells was visualized by staining with antibody against cis-Golgi proteins, GM130 (left, green) and GRASP65 (right, red). Bars, 5 µm. (D) Expression of HA-tagged bovine ARF1 in ARF1-depleted human THP1 cells rescues podosome formation. Podosomes are visualized by phalloidin staining (left) and HA-ARF1 by immunostaining with anti-HA antibody (middle); merged image is shown on the right. Bar, 5 µm. HA-ARF1 was localized to Golgi and to punctate structures shown with high magnification in the right panels representing the enlarged area boxed to the left. Bar, 1 µm. Labeling in the right panel of D shows actin (top), HA-ARF1 (middle), and merged image of both (bottom). Width of the images is 7 µm. (E and F) Quantification of the effect of ARF1 and ARF6 knockdown on podosome integrity. Both number of podosomes per cell (E) and percentage of cells having more than 10 podosomes (F) decreased upon ARF1 but not ARF6 knockdown. Mean ± SD is indicated.This effect was rescued by expression of exogenous HA-ARF1. The graphs represent results of three independent experiments with 100–200 cells used for each group. The numbers of podosomes per cell are presented as a box-and-whiskers plot. Values of median, lower and upper quartiles (box), minimum and maximum values (whiskers) are indicated. The percentages of cells with more than 10 podosomes are presented as mean ± SD. The significance of the difference between groups was estimated by two-tailed Student’s t test. ns, P > 0.05 (nonsignificant); **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.