Figure 1
Figure 1. Two germline mutations in the RUNX1 gene in a familial thrombocytopenia with a propensity to develop acute myeloid leukemia (FPD/AML). (A) Left panels: pedigree A (top) harbors the R174Q; pedigree B (bottom), the R139X mutations. Circles indicate females; squares, males; black filled symbols, affected individuals; gray filled symbol, individual with non hematologic disease; white symbols, nonaffected individuals; and symbols with a diagonal line, deceased individuals. Right panels: analysis of RUNX1 (NM_001001890.2) exon 5 (top panel) showing the heterozygous germline R174Q mutation and exon 4 (bottom panel) showing the heterozygous R139X mutation. (B) Localization of RUNX1 in HEK293T cells after transfection with the lentiviruses encoding for wtRUNX1, or R174Q RUNX1 or R139X RUNX1 and GFP. Immunofluorescence staining of RUNX1 was performed using an anti-HA Ab (red); the nucleus is stained with DAPI (blue). wtRUNX1 protein is localized exclusively in the nucleus with a diffuse pattern. R139X RUNX1 protein is mainly diffused in cytoplasm. R174Q RUNX1 protein is mainly localized in nucleus. In 65% of cells it is localized in dense foci and in 35% of cells, a diffuse pattern is observed. (C) Western blot (WB) analysis of RUNX1, expression in cytoplasmic (C) and nuclear (N) extracts from HEK293T cells overexpressing wild-type (wt), mutant R174Q or mutant R139X RUNX1 (RUNX1tr). RUNX1 forms were tagged with a HA epitope. Western blots (WB) were performed using anti-HA Ab. RUNX1tr: R139X RUNX1 truncated protein. *The nonspecific bands appearing using the anti-HA Ab in nuclear extracts. (D) Interactions between CBFβ and wt or mutant RUNX1 proteins in nuclear extracts from HEK293T cells transfected with CBFβ alone (lane 1) or together with wt RUNX1 (lane 2) or R174Q RUNX1 (lane 3) or R139X RUNX1 (lane 4). CBFβ was tagged with the V5 epitope and RUNX1 forms with a HA epitope. Immunoprecipitation (IP) was performed using an anti-HA Ab and Western blots (WB) using anti-HA and anti-V5 Abs. *Presence of RUNX1tr which is R139X RUNX1 truncated protein.

Two germline mutations in the RUNX1 gene in a familial thrombocytopenia with a propensity to develop acute myeloid leukemia (FPD/AML). (A) Left panels: pedigree A (top) harbors the R174Q; pedigree B (bottom), the R139X mutations. Circles indicate females; squares, males; black filled symbols, affected individuals; gray filled symbol, individual with non hematologic disease; white symbols, nonaffected individuals; and symbols with a diagonal line, deceased individuals. Right panels: analysis of RUNX1 (NM_001001890.2) exon 5 (top panel) showing the heterozygous germline R174Q mutation and exon 4 (bottom panel) showing the heterozygous R139X mutation. (B) Localization of RUNX1 in HEK293T cells after transfection with the lentiviruses encoding for wtRUNX1, or R174Q RUNX1 or R139X RUNX1 and GFP. Immunofluorescence staining of RUNX1 was performed using an anti-HA Ab (red); the nucleus is stained with DAPI (blue). wtRUNX1 protein is localized exclusively in the nucleus with a diffuse pattern. R139X RUNX1 protein is mainly diffused in cytoplasm. R174Q RUNX1 protein is mainly localized in nucleus. In 65% of cells it is localized in dense foci and in 35% of cells, a diffuse pattern is observed. (C) Western blot (WB) analysis of RUNX1, expression in cytoplasmic (C) and nuclear (N) extracts from HEK293T cells overexpressing wild-type (wt), mutant R174Q or mutant R139X RUNX1 (RUNX1tr). RUNX1 forms were tagged with a HA epitope. Western blots (WB) were performed using anti-HA Ab. RUNX1tr: R139X RUNX1 truncated protein. *The nonspecific bands appearing using the anti-HA Ab in nuclear extracts. (D) Interactions between CBFβ and wt or mutant RUNX1 proteins in nuclear extracts from HEK293T cells transfected with CBFβ alone (lane 1) or together with wt RUNX1 (lane 2) or R174Q RUNX1 (lane 3) or R139X RUNX1 (lane 4). CBFβ was tagged with the V5 epitope and RUNX1 forms with a HA epitope. Immunoprecipitation (IP) was performed using an anti-HA Ab and Western blots (WB) using anti-HA and anti-V5 Abs. *Presence of RUNX1tr which is R139X RUNX1 truncated protein.

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