Figure 5.
ESCRT-III complex mediates the function of mDia2. (A) Bone marrow lineage-negative cells from the indicated mice were cultured in erythropoietin-containing medium for 2 days. Immunofluorescent staining of the indicated markers on the orthochromatic erythroblasts was performed on day 2. Bar represents 1 μm. Images are representative of 10 randomly selected fields. (B) Immunofluorescent staining of Chmp5 in the dividing cells of cultured erythroblasts from control and mDia2-deficient mice. Bars represent 1 μm. Images are representative of 10 randomly selected fields. (C) View of the genomic region of Chmp5 with 2 predicated SREs in its promoter region. The sequences of these SREs and their mutant forms used in the luciferase assay are listed. The bottom panel is the luciferase reporter assay. 293T cells were cotransfected with luciferase reporters containing the indicated wild-type (WT) or mutant SREs (MUT), together with SRF-expressing constructs in the presence of a Renilla luciferase expression vector (pRL-TK). Firefly and Renilla luciferase activities were measured after 24 hours. **P < .01. (D) ChIP assay of day 2 cultured wild-type erythroblasts with the indicated antibodies followed by PCR of the SREs on the Chmp5 promoter. (E) ChIP assays as in panel D, using an SRF antibody or IgG on day 2 cultured erythroblasts from the indicated mice followed by PCR of the SREs on the Chmp5 promoter. (F) Real time reverse transcription–PCR analyses of Chmp5 mRNA levels of day 2 cultured erythroblasts from control or mDia2fl/flVav-Cre (knockout) mice transduced with the indicated genes on day 0. ***P < .001. (G) Wild-type bone marrow lineage-negative cells were transduced with murine stem cell virus (MSCV) retroviruses expressing GFP and different shRNAs targeting Chmp5. The transduction efficiency was ∼40% across all the constructs (not shown). The cells were then cultured for 2 days in erythropoietin-containing medium. Immunofluorescent stains of Chmp5 were performed. Images are representative of 5 randomly selected fields. Bars represent 1 μm. (H) Quantification of binucleated orthochromatic erythroblasts in panel G. *P < .05; ***P < .001.

ESCRT-III complex mediates the function of mDia2. (A) Bone marrow lineage-negative cells from the indicated mice were cultured in erythropoietin-containing medium for 2 days. Immunofluorescent staining of the indicated markers on the orthochromatic erythroblasts was performed on day 2. Bar represents 1 μm. Images are representative of 10 randomly selected fields. (B) Immunofluorescent staining of Chmp5 in the dividing cells of cultured erythroblasts from control and mDia2-deficient mice. Bars represent 1 μm. Images are representative of 10 randomly selected fields. (C) View of the genomic region of Chmp5 with 2 predicated SREs in its promoter region. The sequences of these SREs and their mutant forms used in the luciferase assay are listed. The bottom panel is the luciferase reporter assay. 293T cells were cotransfected with luciferase reporters containing the indicated wild-type (WT) or mutant SREs (MUT), together with SRF-expressing constructs in the presence of a Renilla luciferase expression vector (pRL-TK). Firefly and Renilla luciferase activities were measured after 24 hours. **P < .01. (D) ChIP assay of day 2 cultured wild-type erythroblasts with the indicated antibodies followed by PCR of the SREs on the Chmp5 promoter. (E) ChIP assays as in panel D, using an SRF antibody or IgG on day 2 cultured erythroblasts from the indicated mice followed by PCR of the SREs on the Chmp5 promoter. (F) Real time reverse transcription–PCR analyses of Chmp5 mRNA levels of day 2 cultured erythroblasts from control or mDia2fl/flVav-Cre (knockout) mice transduced with the indicated genes on day 0. ***P < .001. (G) Wild-type bone marrow lineage-negative cells were transduced with murine stem cell virus (MSCV) retroviruses expressing GFP and different shRNAs targeting Chmp5. The transduction efficiency was ∼40% across all the constructs (not shown). The cells were then cultured for 2 days in erythropoietin-containing medium. Immunofluorescent stains of Chmp5 were performed. Images are representative of 5 randomly selected fields. Bars represent 1 μm. (H) Quantification of binucleated orthochromatic erythroblasts in panel G. *P < .05; ***P < .001.

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