Transient overexpression of dominant-negative Rab27b mutants, including an Slp1 fragment, results in reduced numbers and length of proplatelet extensions. (A) Quantitative analysis of proplatelet formation (PPF) by fluorescence microscopy 18 to 24 hours following transfection of cultured (day 2.5) primary wild-type MKs using a biolistic “gene-gun” method. The first 2 bars indicate the frequency of PPF among untransfected and mock-transfected MKs under the experimental conditions. There is about a 5-fold decrease in PPF by MKs expressing GFP-tagged Rab27bN133I, Rab27bT23N, or Slp1 (1-150) compared with those expressing wild-type Rab27b. In contrast, MKs expressing GFP-Rab7T22N show a trivial and statistically insignificant decline in PPF. The number of GFP⁺ cells scored for each construct and the statistical significance (Student t test) of the numeric differences between wild-type (WT) and dominant-negative samples are indicated above the histogram bars. Data (shown as mean ± SD) are pooled from 3 to 6 independent experiments (for the different constructs), which gave consistent results. (B) Representative fluorescence micrographs (original magnification, × 200) of proplatelet-forming MKs reveal the significant shortening of proplatelet shafts in cells expressing GFP-tagged Rab27bN133I or Slp1(1-150). Cells transfected with the wild-type or dominant inhibitory constructs are deliberately shown at different magnifications (original magnifications × 1 vs × 5) to highlight the appearance of proplatelets observed in each case.