Figure 1.
CRISPR–mediated GLUT1 KO on immortalized BEL-A erythroblasts successfully generate reticulocytes. (A) Sequencing of SLC2A1 (GLUT1) gene on Ctrl BEL-A, highlighting the guide RNA (green) used for CRISPR editing. Sanger sequencing of clonal edited lines shows a homozygous 4–base pair (bp) frameshift mutation on the GLUT1 KO line, and a heterozygotic 16-bp deletion on the KD line, both in the vicinity of the cutting site (red line). Flow cytometry histogram of GLUT1 staining in BEL-A erythroblasts (B) and derived reticulocytes (D) from Ctrl (green), GLUT1 KD (blue), and GLUT1 KO (orange) cell lines compared with no-stain control (red). Cells were stained with anti-GLUT1 eGFP conjugate. (C) Flow cytometry analysis of cell surface marker expression during differentiation. Cells were colabeled with anti-band3 primary antibody used in conjunction with an immunoglobulin G1 (IgG1) APC secondary and anti–α4-integrin FITC conjugate. For day 11, reticulocytes were identified using Hoechst as a nuclear DNA stain. (E) Bar graph illustrates the percentage GLUT1 expression on reticulocytes derived from indicated cell lines. Data are normalized to endogenous expression of Ctrl BEL-A from the median fluorescence intensity (n = 4). Individual data points are shown. Error bars indicate standard error of mean. (F) Representative images of May-Grünwald and Giemsa–stained cytospins depicting expanding BEL-A erythroblasts (day 0) and corresponding filtered reticulocytes after 10 day differentiation protocol; 40× original magnification. Scale bars, 20 μm, shown for each image. (G) Bar graphs illustrate expression of various membrane proteins on reticulocytes derived from indicated cell lines (n = 3). Reticulocytes were identified based on Hoechst stain negativity. Data are normalized to endogenous expression of Ctrl BEL-A and represents the median fluorescence intensity (n = 3). Individual data points are shown. Error bars indicate standard error of mean. (H) Western blots of lysates obtained from indicated cell lines at day 0 of differentiation and reticulocytes filtered after 10-day protocol, incubated with antibodies to α-adducin, GLUT1, stomatin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control). Multiple Mann-Whitney U tests were used to test for differences between groups. ∗P < .05. Error bars indicate standard deviation.

CRISPR–mediated GLUT1 KO on immortalized BEL-A erythroblasts successfully generate reticulocytes. (A) Sequencing of SLC2A1 (GLUT1) gene on Ctrl BEL-A, highlighting the guide RNA (green) used for CRISPR editing. Sanger sequencing of clonal edited lines shows a homozygous 4–base pair (bp) frameshift mutation on the GLUT1 KO line, and a heterozygotic 16-bp deletion on the KD line, both in the vicinity of the cutting site (red line). Flow cytometry histogram of GLUT1 staining in BEL-A erythroblasts (B) and derived reticulocytes (D) from Ctrl (green), GLUT1 KD (blue), and GLUT1 KO (orange) cell lines compared with no-stain control (red). Cells were stained with anti-GLUT1 eGFP conjugate. (C) Flow cytometry analysis of cell surface marker expression during differentiation. Cells were colabeled with anti-band3 primary antibody used in conjunction with an immunoglobulin G1 (IgG1) APC secondary and anti–α4-integrin FITC conjugate. For day 11, reticulocytes were identified using Hoechst as a nuclear DNA stain. (E) Bar graph illustrates the percentage GLUT1 expression on reticulocytes derived from indicated cell lines. Data are normalized to endogenous expression of Ctrl BEL-A from the median fluorescence intensity (n = 4). Individual data points are shown. Error bars indicate standard error of mean. (F) Representative images of May-Grünwald and Giemsa–stained cytospins depicting expanding BEL-A erythroblasts (day 0) and corresponding filtered reticulocytes after 10 day differentiation protocol; 40× original magnification. Scale bars, 20 μm, shown for each image. (G) Bar graphs illustrate expression of various membrane proteins on reticulocytes derived from indicated cell lines (n = 3). Reticulocytes were identified based on Hoechst stain negativity. Data are normalized to endogenous expression of Ctrl BEL-A and represents the median fluorescence intensity (n = 3). Individual data points are shown. Error bars indicate standard error of mean. (H) Western blots of lysates obtained from indicated cell lines at day 0 of differentiation and reticulocytes filtered after 10-day protocol, incubated with antibodies to α-adducin, GLUT1, stomatin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control). Multiple Mann-Whitney U tests were used to test for differences between groups. ∗P < .05. Error bars indicate standard deviation.

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