Figure 4.

Subjects have a point mutation in KINDLIN3. (a) Western blot for KINDLIN-3 in subjects’ and control lymphocytes. The positions of full-length KINDLIN-3 and its short form are indicated with arrows. Lanes 1 and 2 show samples of immortalized cell lines from different healthy individuals; lanes 3 and 4 show subjects 1 and 2, respectively. (b) Chromatograms of genomic PCR sequence. Color-coded nucleotide sequence is shown at the bottom. Analyses of subjects 1 and 2 as well as their parents (M and F) are shown. Arrow indicates the point mutation. (c) KINDLIN3 expression rescues adhesion and spreading defects in subject 1’s cells. Cells from subject 1, a healthy control and the K562 cell line were transfected with KINDLIN3-EGFP or EGFP vector control as indicated, and equal amounts of cells were plated on a fibrinogen-coated plate as described in the Supplementary Methods. Adherent cells were counted in six random fields. The data represent means ± s.d., n = 3, ** P < 0.01. (d) Expression of KINDLIN3 rescues the defect in spreading on fibronectin in subject 1’s cells. Microphotographs of EGFP- or KINDLIN3-EGFP–transfected subject 1’s cells are shown. Left, Nomarski microscopy (DIC, differential interference contrast); right, fluorescence microscopy (FL). Scale bar, 10 μm. (e) TIRF analysis of KINDLIN3-EGFP–expressing subject 1’s cells adhered to fibrinogen-coated plates. Cells were transfected with EGFP or KINDLIN3-EGFP as indicated. Cells were visualized using TIRF (left), Nomarski (center) or fluorescence (right) microscopy. Scale bar, 10 μm. (f) Knockdown of KINDLIN3 in control cells recapitulates the subjects’ cells’ adhesion and spreading defects. K562 lymphocytes or control cell lines were transfected with KINDLIN3–specific siRNA, control siRNA or remained untransfected as indicated. Adhesion assays were performed as in d. The data represent means ± s.d., n = 3, ** P < 0.01.