Properties of CD34+ GLUT1-KO–derived reticulocytes. (A) Waterfall plot indicating progression of control and GLUT1-KO–transfected cultures of single donor CD34+ differentiation. Cells were colabeled with anti-band3 primary antibody used in conjunction with an IgG1 APC secondary and anti–α4-integrin FITC conjugate. For day 20, reticulocytes were identified using Hoechst stain as a nuclear DNA stain. (B) Representative cytospins of the same culture were obtained on day 20 after leukofiltration. 40× original magnification. Scale bars, 20 μm, shown for each image. (C) Bar graphs indicate the expression of various membrane proteins on reticulocytes derived from NT or GLUT1-KO primary cultures (n = 6, for SMVT n = 3, with 2 technical repeats each). Significance was assessed by multiple Mann-Whitney U tests with a false discovery rate of 1% to account for multiple comparisons. “∗” shows q < .01 and “nd” shows q > .01. (D) Anti-transferrin receptor (CD71) labeling of on reticulocytes (n = 6, open or filled dots indicate 2 separate cultures, each with 3 donors). For both, reticulocytes were leukofiltered on day 20. Data are normalized to each donor-matched NT control and represents the median fluorescence intensity. (E) Osmotic resistance analysis calculated based on viable cell counts (flow cytometer) after incubation with decreasing concentrations of NaCl (n = 3, 2 technical replicates, “∘” P ≤ .0021). (F) Deformability and (G) cell area (μm2) were measured under shear stress by automated rheoscope cell analyzer (n = 3, N > 2000 cells), which elongates cells and measures length over width as deformability parameter. Shaded region represents the standard deviation. (H) Quantitative analysis of lipid peroxidation detected by a shift in the fluorescence signal after treatment with 25 mM cumene hydroperoxide. Data normalized to each donor-matched NT control (n = 3, 3 technical replicates). (I) Bar graph quantifying P falciparum reticulocyte invasion efficiency. Invasion was assessed by flow cytometry using a SYBR-green DNA stain (3 separate parasitemia percentages, 3 technical replicates) and data were normalized per matched-donor (n = 3) NT control. This figure comprises data obtained from 2 independent cultures, each of 3 donors with panels A-B presenting representative data from cultures with fluorescence-activated cell sorted GLUT1-KO purity of 99%; panels E-I 94% and panels C-D combined data from all 6 donors. A nonparametric Mann-Whitney U test or Kruskal-Wallis test with Bonferroni correction were used to test for differences between groups when not specifically mentioned, ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. Error bars indicate standard deviation.
