PSTPIP1 impairs podosome formation. (A-B) Lentiviral transduction of shRNA was used to knock down PSTPIP1 in THP-1 cells. (A) Western blot of lysates from the control (shCtrl) and PSTPIP1 knockdown (shPSTPIP1 or shPST) THP-1 cells using anti-PSTPIP1 and anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (loading control). Arrowheads indicate location of molecular weight markers. (B) Quantification of PSTPIP1 knockdown in which PSTPIP1 expression in shCtrl cells was normalized to 100%. (C) Representative images of podosomes in shCtrl and shPSTPIP1 THP-1 macrophages. Podosomes were stained with anti-vinculin antibody (green) and rhodamine phalloidin (magenta). (D) Quantification of the average number of podosomes formed by shCtrl and shPSTPIP1 THP-1 cells. (E) Representative images of gelatin degradation by PSTPIP1 knockdown cells on Alexa Fluor 568–conjugated gelatin (magenta) coverslips. Podosomes were stained with Alexa Fluor 488 phalloidin (green). The cell outline is a trace of the cell border made from the phalloidin image. Orange pixels in the Degraded Area panel were those counted as degraded. (F) Quantification of the amount of gelatin degraded by PSTPIP1 knockdown cells as a percentage of cell area. (G) Cell area was determined in all cells for the degradation experiment and normalized to the shCtrl value. Scale bars, 20 μm. All values are mean ± standard error of the mean (SEM) from 3 (D,F,G) or 4 (B) independent experiments. *P < .05; ***P < .001 as determined by a paired Student t test (B,D,G) or ANOVA with repeated measures using a compound symmetry correlation structure (F). NS, nonsignificant.