Figure 5
Altered expression of DIAPH1-3 and cytoskeletal organization in platelets from DIAPH1 R1213* variant cases. (A) Representative western blots of resolved platelet protein extracts from DIAPH1 R1213* cases 10, 16, and 17 and from a control probed with antibodies recognizing DIAPH1, DIAPH3, DIAPH2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compared with the control, the DIAPH1 R1213* cases show normal expression of DIAPH1. The content of DIAPH2 and DIAPH3 is increased in the cases compared with control. Similar quantities of total protein in the western blot lanes are indicated by the control blot probed with an antibody recognizing GAPDH. (B) Representative confocal microscopy images of poly-l-lysine-immobilized resting platelets from cases 10 and 16 and from a control stained for DIAPH1 (cyan), F-actin (red), and α-tubulin (green). Platelets were visualized using a Leica TCS SP5 confocal microscope (Leica Microsystems). Bars represent 3 μm. (C) Image analysis (ratio of the mean of the first and last maxima and the mean of the first and last minima) revealed an aberrant distribution of DIAPH1 in platelets from cases 10 and 16 as compared with controls. Box plots display first and third quartiles, and whiskers mark minimum and maximum values unless exceeding 1.5 times the interquartile range of at least 50 platelets per group; symbols represent outliers, and the horizontal line displays the median. Wilcoxon-Mann-Whitney test, ***P < .001. (D-E) Quantification of the immunostained α-tubulin surface (D) and fluorescence intensity per surface unit of the F-actin staining (E) revealed an increased content and an abnormal distribution in platelets from the cases. Values represent means ± SD (n = 3 controls vs case 10 and 16; 100 platelets). Wilcoxon-Mann-Whitney test, ***P < .001. (F) Representative transmission electron micrographs showing that the microtubules (indicated by arrows) are disorganized and distributed throughout the cytoplasm of platelets from case 10 compared with controls in which microtubules are organized into the marginal band. Images were collected using an EM900 (Carl Zeiss) electron microscope. Bars represent 0.5 μm. (G) Manual counting of microtubules revealed an increased number of microtubules in platelets from the cases (n = 41 platelets) compared with controls (n = 104 platelets). Microtubule numbers per platelet are expressed as mean ± SD. Unpaired Student t test, ***P < .001. AU, arbitrary units; MFI, mean fluorescence intensity; MT, microtubule; Rel. FI, relative fluorescence intensity; SD, standard deviation.

Altered expression of DIAPH1-3 and cytoskeletal organization in platelets from DIAPH1 R1213* variant cases. (A) Representative western blots of resolved platelet protein extracts from DIAPH1 R1213* cases 10, 16, and 17 and from a control probed with antibodies recognizing DIAPH1, DIAPH3, DIAPH2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compared with the control, the DIAPH1 R1213* cases show normal expression of DIAPH1. The content of DIAPH2 and DIAPH3 is increased in the cases compared with control. Similar quantities of total protein in the western blot lanes are indicated by the control blot probed with an antibody recognizing GAPDH. (B) Representative confocal microscopy images of poly-l-lysine-immobilized resting platelets from cases 10 and 16 and from a control stained for DIAPH1 (cyan), F-actin (red), and α-tubulin (green). Platelets were visualized using a Leica TCS SP5 confocal microscope (Leica Microsystems). Bars represent 3 μm. (C) Image analysis (ratio of the mean of the first and last maxima and the mean of the first and last minima) revealed an aberrant distribution of DIAPH1 in platelets from cases 10 and 16 as compared with controls. Box plots display first and third quartiles, and whiskers mark minimum and maximum values unless exceeding 1.5 times the interquartile range of at least 50 platelets per group; symbols represent outliers, and the horizontal line displays the median. Wilcoxon-Mann-Whitney test, ***P < .001. (D-E) Quantification of the immunostained α-tubulin surface (D) and fluorescence intensity per surface unit of the F-actin staining (E) revealed an increased content and an abnormal distribution in platelets from the cases. Values represent means ± SD (n = 3 controls vs case 10 and 16; 100 platelets). Wilcoxon-Mann-Whitney test, ***P < .001. (F) Representative transmission electron micrographs showing that the microtubules (indicated by arrows) are disorganized and distributed throughout the cytoplasm of platelets from case 10 compared with controls in which microtubules are organized into the marginal band. Images were collected using an EM900 (Carl Zeiss) electron microscope. Bars represent 0.5 μm. (G) Manual counting of microtubules revealed an increased number of microtubules in platelets from the cases (n = 41 platelets) compared with controls (n = 104 platelets). Microtubule numbers per platelet are expressed as mean ± SD. Unpaired Student t test, ***P < .001. AU, arbitrary units; MFI, mean fluorescence intensity; MT, microtubule; Rel. FI, relative fluorescence intensity; SD, standard deviation.

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