Knockdown of ARF1 exchange factor ARNO (cytohesin 2), but not cytohesin 1, leads to podosome disruption. (A) Western blot showing ARNO and cytohesin 1 levels in cells treated with control (scrambled) siRNA, ARNO, or cytohesin 1 siRNAs; α-tubulin was used as a loading control. (B, top) TGFβ1-stimulated THP1 cells transfected with scrambled siRNAs; podosomes labeled with phalloidin and Golgi apparatus labeled by antibody against GM130. (middle) siRNA-mediated knockdown of ARNO disrupted podosomes, leaving the Golgi undisturbed. (bottom) Cytohesin 1 knockdown disrupted neither podosomes nor Golgi. Bars, 5 µm. (C and D) Quantification of the effect of ARNO and cytohesin 1 knockdown on number of podosomes per cell (C) and percentage of cells with more than 10 podosomes (D). (E) G-LISA quantification of ARF1-GTP level in nontransfected TGFβ1-stimulated THP1 cells (control), scrambled siRNA–transfected cells, and cells transfected with ARNO siRNA. Mean ± SD is indicated. (F and G) Quantification of the effect of expression of wild-type GFP-ARNO and dominant-negative ARNO mutant (GFP–ARNO E156K) on mean number of podosomes per cell (F) and percentage of cells with more than 10 podosomes (G). The data in C, D, F, and G 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 ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.