American Society of Hematology

Figure 3

$Figure 3. Erythroid differentiation is blocked in Mbnl1 knockdown cells and not restored by ectopic expression of the excluded isoform. (A) Western blot analysis of MBNL1 protein from GFP-sorted day 2 cultured cells following expression of shLuc, shMbnl1-1, and shMbnl1-2. GAPDH was used as loading control. (B) May-Grunwald Giemsa staining of GFP-sorted day 2 cultured cells infected with the indicated viruses. Representative images are shown. Scale bar is 10 µm. (C) Cell size measured by forward scatter at day 2 of in vitro culture shown for cells expressing shLuc control (red), shMbnl1-1 (green), and shMbnl1-2 (blue). Bar graph shows the relative forward scatter level of cells treated with the shLuc or shMbnl1 shRNAs. Statistics from 3 experiments are shown. (D) Flow cytometry plots of day 2 in vitro cultured cells stained with Ter119-APC and Hoechst. Boxes denote enucleated cells. Statistics from 3 experiments are shown. (E) RNA expression levels of the indicated genes in GFP-sorted day 2 cultured cells following addition of shRNAs, as determined by qRT PCR. 18s rRNA was used for normalization. Error bar is standard deviation (n = 3). (F) Relative hemoglobin levels in GFP-sorted day 2 cultured cells following addition of the indicated shRNAs (n = 3). (G) Western blot analysis of Mbnl1 subcellular localization in E14.5 murine fetal liver cells. GAPDH and hemoglobin α served as markers for the cytoplasmic fraction, whereas histone 2A.x served as marker for the nuclear fraction. (H) qPCR analysis of the expression levels of the Mbnl1 inclusion and exclusion isoforms after knockdown of the Mbnl1 inclusion isoform. (I) Terminal proliferation of erythroid cells after knockdown of the Mbnl1 inclusion isoform. (J) Cell size of erythroid cells measured by forward scatter after knockdown of the Mbnl1 inclusion and exclusion isoforms. (K) Enucleation of erythroid cells after knockdown of the Mbnl1inclusion and excluded isoforms. (L) qPCR analysis of the expression levels of splicing isoforms of 4 genes after knockdown of the Mbnl1 inclusion and exclusion isoforms. (M) qPCR analysis of the expression levels of the Mbnl1 inclusion and exclusion isoforms after knockdown of the Mbnl1inclusion isoform with or without ectopic expression of the Mbnl1 exclusion isoform. (N) Cell size, (O) enucleation, and (P) qPCR analysis of the expression levels of splicing isoforms of 4 genes of erythroid cells after knockdown of the Mbnl1inclusion isoform with or without ectopic expression of the Mbnl1 exclusion isoform.$

Erythroid differentiation is blocked in Mbnl1 knockdown cells and not restored by ectopic expression of the excluded isoform. (A) Western blot analysis of MBNL1 protein from GFP-sorted day 2 cultured cells following expression of shLuc, shMbnl1-1, and shMbnl1-2. GAPDH was used as loading control. (B) May-Grunwald Giemsa staining of GFP-sorted day 2 cultured cells infected with the indicated viruses. Representative images are shown. Scale bar is 10 µm. (C) Cell size measured by forward scatter at day 2 of in vitro culture shown for cells expressing shLuc control (red), shMbnl1-1 (green), and shMbnl1-2 (blue). Bar graph shows the relative forward scatter level of cells treated with the shLuc or shMbnl1 shRNAs. Statistics from 3 experiments are shown. (D) Flow cytometry plots of day 2 in vitro cultured cells stained with Ter119-APC and Hoechst. Boxes denote enucleated cells. Statistics from 3 experiments are shown. (E) RNA expression levels of the indicated genes in GFP-sorted day 2 cultured cells following addition of shRNAs, as determined by qRT PCR. 18s rRNA was used for normalization. Error bar is standard deviation (n = 3). (F) Relative hemoglobin levels in GFP-sorted day 2 cultured cells following addition of the indicated shRNAs (n = 3). (G) Western blot analysis of Mbnl1 subcellular localization in E14.5 murine fetal liver cells. GAPDH and hemoglobin α served as markers for the cytoplasmic fraction, whereas histone 2A.x served as marker for the nuclear fraction. (H) qPCR analysis of the expression levels of the Mbnl1 inclusion and exclusion isoforms after knockdown of the Mbnl1 inclusion isoform. (I) Terminal proliferation of erythroid cells after knockdown of the Mbnl1 inclusion isoform. (J) Cell size of erythroid cells measured by forward scatter after knockdown of the Mbnl1 inclusion and exclusion isoforms. (K) Enucleation of erythroid cells after knockdown of the Mbnl1inclusion and excluded isoforms. (L) qPCR analysis of the expression levels of splicing isoforms of 4 genes after knockdown of the Mbnl1 inclusion and exclusion isoforms. (M) qPCR analysis of the expression levels of the Mbnl1 inclusion and exclusion isoforms after knockdown of the Mbnl1inclusion isoform with or without ectopic expression of the Mbnl1 exclusion isoform. (N) Cell size, (O) enucleation, and (P) qPCR analysis of the expression levels of splicing isoforms of 4 genes of erythroid cells after knockdown of the Mbnl1inclusion isoform with or without ectopic expression of the Mbnl1 exclusion isoform.

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