Figure 5.
MKs from probands carrying GALE variants displayed impaired GPIbα and β1 glycosylation and externalization. MKs were differentiated from peripheral blood progenitor cells (probands A.II.4 and B.II.2) through a 14-days culture, in parallel with healthy controls. (A) MFI of β3 integrin (anti-CD61), GPIbα (anti-CD42b), and β1 integrin (anti-CD29) in permeabilized MKs (total levels) and nonpermeabilized MKs (surface levels) from controls, A.II.4, and B.II.2 are represented. Data were normalized against 1 control value. (B) Representative analysis of the automized immunoblotting of GPIbα and Western blotting of β1 integrin from a healthy control and proband B.II.2. Red dotted lines highlight bands of lower molecular weight in MKs from patients. Densitometry analysis demonstrated comparable levels of total proteins between patients and controls. β3 integrin was used as an internal control. (C) Representative image of control, A.II.4, and B.II.2 MKs. Cells were cytospinned and stained with anti-GPIbα or anti-β1–integrin antibodies (green). The ER was stained with anti-calreticulin (red fluorescence). Hoechst (blue) was used for counterstaining nuclei. Pearson’s R values indicate the colocalization rate between GPIbα or β1 integrin with calreticulin in control and probands A.II.4 and B.II.2 MKs. (D) Fluorescence intensity distribution of GPIbα and β1 integrin in control vs patients. Control MKs presented a major distribution of GPIbα and β1 integrin in the membrane, with reduced intracellular levels, whereas patients’ fluorescence distribution was mainly intracellular. Scale bars, 10 μm. ∗∗∗P < .001; ∗∗P < .01; 2-tailed Student t test. CALR, calreticulin.

MKs from probands carrying GALE variants displayed impaired GPIbα and β1 glycosylation and externalization. MKs were differentiated from peripheral blood progenitor cells (probands A.II.4 and B.II.2) through a 14-days culture, in parallel with healthy controls. (A) MFI of β3 integrin (anti-CD61), GPIbα (anti-CD42b), and β1 integrin (anti-CD29) in permeabilized MKs (total levels) and nonpermeabilized MKs (surface levels) from controls, A.II.4, and B.II.2 are represented. Data were normalized against 1 control value. (B) Representative analysis of the automized immunoblotting of GPIbα and Western blotting of β1 integrin from a healthy control and proband B.II.2. Red dotted lines highlight bands of lower molecular weight in MKs from patients. Densitometry analysis demonstrated comparable levels of total proteins between patients and controls. β3 integrin was used as an internal control. (C) Representative image of control, A.II.4, and B.II.2 MKs. Cells were cytospinned and stained with anti-GPIbα or anti-β1–integrin antibodies (green). The ER was stained with anti-calreticulin (red fluorescence). Hoechst (blue) was used for counterstaining nuclei. Pearson’s R values indicate the colocalization rate between GPIbα or β1 integrin with calreticulin in control and probands A.II.4 and B.II.2 MKs. (D) Fluorescence intensity distribution of GPIbα and β1 integrin in control vs patients. Control MKs presented a major distribution of GPIbα and β1 integrin in the membrane, with reduced intracellular levels, whereas patients’ fluorescence distribution was mainly intracellular. Scale bars, 10 μm. ∗∗∗P < .001; ∗∗P < .01; 2-tailed Student t test. CALR, calreticulin.

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