Figure 3.
Figure 3. Wright-Giemsa staining, PS exposure, intracellular calcium, sensitivity to hyperosmolarity, and osmotic fragility of HNF1A−/−RBCs. (A) Representative light microscopy images of Wright-Giemsa–stained peripheral blood smears (n = 3; original magnification ×100) of HNF1A+/+ and HNF1A−/− mice. Bars represent 10 μm. (B) Representative images of annexin V flow cytometric analysis of HNF1A+/+ (n = 6) and HNF1A−/− (n = 6) RBCs, 16 hours after collecting peripheral blood samples and incubation at room temperature. The FL2 channel was empty (i). Quantification of the mean percentage of annexin V binding on HNF1A+/+ and HNF1A−/− RBCs (n = 6 each) 16 hours after collecting blood samples (ii) or 6 hours after glucose deprivation of washed RBCs (iii). **P < .01 and ***P < .001 (Student t test). (C) Representative flow cytometric analysis of intracellular calcium content in HNF1A+/+ and HNF1A−/− RBCs prestained with Fluo-4 and incubated in Ringer without (i, top) and with ionomycin (1 µM; i, middle), with ionomycin (1 µM; i, bottom) in Ringer without Ca2+ in the presence of 1 mM EDTA measured after 2 minutes of incubation. The FL2 channel was empty. Quantification of the percentage (ii) and mean fluorescence intensity (MFI) (iii) of HNF1A+/+ (n = 4) and HNF1A−/− (n = 4) RBCs positive for Fluo-4. **P < .01 and ***P < .001 for untreated HNF1A+/+ vs HNF1A−/−, †††P < .001 for treated vs untreated HNF1A+/+ and §§P < .01 and §§§P < .001 for treated vs untreated HNF1A−/− (1-way ANOVA followed by Tukey post hoc analysis). (D) Mean percentage of annexin V binding on HNF1A+/+ and HNF1A−/− RBCs (n = 3 each) 4 hours after incubation with 950 mOsm Ringer solution in the presence or absence of 50 µM amitriptyline (Ami). *P < .05, **P < .01, and ***P < .001 for 950 mOsm vs untreated HNF1A+/+ or vs 950 mOsm plus Ami; for 950 mOsm vs untreated HNF1A−/− or vs 950 mOsm plus Ami as indicated (1-way ANOVA followed by Tukey post hoc analysis). (E) Osmotic fragility in RBCs from HNF1A+/+ and HNF1A−/− mice (n = 3 each) using solutions with decreasing concentrations of NaCl. **P < .01 and ***P < .001 for untreated vs treated RBCs (2-way ANOVA followed by Bonferroni post hoc analysis). ns, not significant.

Wright-Giemsa staining, PS exposure, intracellular calcium, sensitivity to hyperosmolarity, and osmotic fragility of HNF1A−/−RBCs. (A) Representative light microscopy images of Wright-Giemsa–stained peripheral blood smears (n = 3; original magnification ×100) of HNF1A+/+ and HNF1A−/− mice. Bars represent 10 μm. (B) Representative images of annexin V flow cytometric analysis of HNF1A+/+ (n = 6) and HNF1A−/− (n = 6) RBCs, 16 hours after collecting peripheral blood samples and incubation at room temperature. The FL2 channel was empty (i). Quantification of the mean percentage of annexin V binding on HNF1A+/+ and HNF1A−/− RBCs (n = 6 each) 16 hours after collecting blood samples (ii) or 6 hours after glucose deprivation of washed RBCs (iii). **P < .01 and ***P < .001 (Student t test). (C) Representative flow cytometric analysis of intracellular calcium content in HNF1A+/+ and HNF1A−/− RBCs prestained with Fluo-4 and incubated in Ringer without (i, top) and with ionomycin (1 µM; i, middle), with ionomycin (1 µM; i, bottom) in Ringer without Ca2+ in the presence of 1 mM EDTA measured after 2 minutes of incubation. The FL2 channel was empty. Quantification of the percentage (ii) and mean fluorescence intensity (MFI) (iii) of HNF1A+/+ (n = 4) and HNF1A−/− (n = 4) RBCs positive for Fluo-4. **P < .01 and ***P < .001 for untreated HNF1A+/+ vs HNF1A−/−, †††P < .001 for treated vs untreated HNF1A+/+ and §§P < .01 and §§§P < .001 for treated vs untreated HNF1A−/− (1-way ANOVA followed by Tukey post hoc analysis). (D) Mean percentage of annexin V binding on HNF1A+/+ and HNF1A−/− RBCs (n = 3 each) 4 hours after incubation with 950 mOsm Ringer solution in the presence or absence of 50 µM amitriptyline (Ami). *P < .05, **P < .01, and ***P < .001 for 950 mOsm vs untreated HNF1A+/+ or vs 950 mOsm plus Ami; for 950 mOsm vs untreated HNF1A−/− or vs 950 mOsm plus Ami as indicated (1-way ANOVA followed by Tukey post hoc analysis). (E) Osmotic fragility in RBCs from HNF1A+/+ and HNF1A−/− mice (n = 3 each) using solutions with decreasing concentrations of NaCl. **P < .01 and ***P < .001 for untreated vs treated RBCs (2-way ANOVA followed by Bonferroni post hoc analysis). ns, not significant.

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