PMC6759462_01
PMC6759462_02
(A) The results of hematological testing and infectious diseases during the course of the disease. (B) The results of immunological testing during the course of the disease. BM, bone marrow; CMV, cytomegalovirus; IFNg, interferon gamma; SCT, hematopoietic stem cell transplantation.
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F1
Figure 1B. Flow cytometry (FC) determination (first performed at the age of 5 months) of major lymphocyte subsets did not provide conclusive results.
PMC6759462_03
PMC6759462_04
(A) The results of hematological testing and infectious diseases during the course of the disease. (B) The results of immunological testing during the course of the disease. BM, bone marrow; CMV, cytomegalovirus; IFNg, interferon gamma; SCT, hematopoietic stem cell transplantation.
fimmu-10-02194-g0001.jpg
PMC6759462_04_fimmu-10-02194-g0001.jpg
F1
Figure 1B. Flow cytometry (FC) determination (first performed at the age of 5 months) of major lymphocyte subsets did not provide conclusive results.
(A) T-cell culture viability determined by flow cytometry. Live cells were gated based on propidium iodide and forward scatter signals. (B) Western blot analysis of Bcl-2 and Bcl-xL expression in T cells after 2, 3, and 5 weeks of culture. Ctrl, control, Pt, patient. (C) Flow cytometry analysis of T-cell calcium response to activation with 1 and 100 mug/ml anti-CD3epsilon antibody. The arrow indicates the time point when antibody was added to the sample.
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F2
T-cell cultures were established by stimulating PBMCs with immobilized anti-CD3epsilon antibody followed by propagation in the presence of IL-2. These cultures displayed reduced viability, accompanied by the lack of anti-apoptotic Bcl-2 protein expression (Figures 2A,B). Upon CD3 re-stimulation, they showed slightly decreased calcium flux (Figure 2C), while no difference in overall tyrosine phosphorylation after TCR stimulation was observed (data not shown).
Analysis of proliferation (A), cell death (B) of HEK293T cells transfected with wt SAMD9-GFP, mutant SAMD9-GFP or empty GFP-positive control expression vector. Representative data are shown with results depicted as a fraction of GFP+ cells. Statistical significance: (*P <= 0.05; **P <= 0.001; ***P <= 0.0001).
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F3
The impact of mutant SAMD9 was even more profound resulting in a dramatic drop in the number of proliferating cells (Figure 3A). Interestingly, mutant SAMD9 induced primary necrosis of the cultured cells further demonstrating the gain-of-function impact of p.R824Q on SAMD9 (Figure 3B).