Fig. 3
From: Variants in exons 5 and 6 of ACTB cause syndromic thrombocytopenia
Reduced cell attachment surface area, volume and migratory capacity of ACTB-AST fibroblasts. a Micrographs of control (C), patient 4 (P4, p.Ala331Valfs*27) and patient 5 (P5, p.Ser338_Ile341del) primary dermal fibroblasts at high (top row) and low (bottom row) confluence. At low confluence, ACTB-AST cells are distinctly smaller than controls and P5 cells grow in aggregates (arrow). All scale bars are 100 µm; b Quantification of the cell attachment surface area from immunofluorescence analyses (i.e. Figure 4e) shows reduced coverage distribution by ACTB-AST cells (median and IQR, the number of cells analyzed in 4 experiments is given in brackets); c Flow cytometry analysis of the Forward Scatter Area (FSC-A) vs. normalized cell count (100,000 events from 1 experiment) shows a reduction in ACTB-AST cell volume (P4: pink, P5: purple) compared to the control (green); d–g Migration assays demonstrate reduced migratory capacity for ACTB-AST patient primary fibroblasts; d Representative images at 8 h with migratory tracks overlaid. Scale bars are 100 µm; e Migration speed of individual cells represented in µm per hour (median and IQR); f Trajectories of all tracks recorded for C, P4 and P5 from 0 h (origin) to 8 h (5 movies from 2 technical replicates, n = number of cells analyzed); g Mean square displacement analysis of C (green), P4 (magenta) and P5 (purple) fibroblasts (mean ± s.e.m.). Significance was determined with the Kruskal–Wallis test, where ***p < 0.001 and ****p < 0.0001
