Overexpression of DIAPH1 R1213* in cell lines reproduces the cytoskeletal alterations in platelets. (A) Western blot of protein extracts from HEK293FT cells transfected with DIAPH1 WT (WT), DIAPH1 R1213* (R1213*), or empty (C) expression constructs, probed with antibodies recognizing the DIAPH1 amino terminus, DIAPH3, or DIAPH2. Confocal microscopy images of A549 cells transiently transfected with the DIAPH1 WT or R1213* expression constructs were stained for F-actin (red), α-tubulin (green), and DIAPH1 (cyan) (B) and for F-actin (red), ac-tub (green), and DIAPH1 (cyan) (C) and with DAPI nuclear counterstain (blue) (B-C). (D) Quantification of the relative fluorescence intensity per surface unit of transfected and nontransfected cells revealed an increased content of F-actin, α-tubulin, and ac-tub in the cells overexpressing DIAPH1 R1213* compared with adjacent nontransfected cells (differences are indicated by asterisks) and DIAPH1 WT–overexpressing cells (differences are indicated by pound signs). (E-F) Incubation with the FH2 domain inhibitor SMIFH2 reduced F-actin content, but not the content of microtubules and ac-tub as determined by quantification of the relative fluorescence intensity per surface unit. Values in panels D and F are expressed as means ± SD (n = 100 cells). Wilcoxon-Mann-Whitney test, **^,##P < .01; ***^,###P < .001. The cells in panels B, C, and E were visualized using a Leica TCS SP5 confocal microscope (Leica Microsystems). Bars represent 10 μm.