PSTPIP1-R405C promotes filopodia formation. (A) Diagram of constructs introduced into control and PSTPIP1 knockdown cells by retroviral transduction to make rescue cell lines. (B) Representative western blot of lysates from FACS-sorted THP-1 rescue lines showing expression of rescue constructs. Blots were probed with mouse anti-PSTPIP1, rabbit anti-GFP, and mouse anti-GAPDH (loading control) antibodies. Arrowheads indicate location of molecular weight markers. (C) Representative images of podosomes and filopodia formed by the THP-1 rescue cell lines, which were stained with anti-vinculin antibody (green) and rhodamine phalloidin (magenta). Anti-GFP antibody (green) was used to amplify the GFP signal. Inset shows filopodia at ×1.5 original magnification. (D) Slices through the Z-plane of the shPST + WT and shPST + R405C cells were created with the Reslice function in FIJI software as indicated by the dashed lines in (C). (E) Quantification of the average number of podosomes formed by the indicated rescue line. shCtrl + GFP was the reference value for statistical comparison except where indicated by a bar. (F) Quantification of the percentage of shPST + WT and shPST + R405C cells that form filopodia. Scale bars, 20 μm. All values are mean ± SEM from 3 independent experiments. *P < .05; **P < .01 as determined by ANOVA with repeated measures using a compound symmetry correlation structure (E) or paired Student t test (F). NS, nonsignificant.