Figure 6.
Expression of cIL-4Rα ameliorates macrothrombocytopenia. (A) Anticoagulated whole blood from and hGPIbαFW and hGPIbαFW/cIL-4Rα mice was assessed for platelet GPIbα surface expression by flow cytometry using anti-CD42b Ab-PE. The results are the mean fluorescence intensity of gated platelet population (mean ± SEM of n = 10 pairs hGPIbαFW and hGPIbαFW/cIL-4Rα mice). (B-C) Platelet count (B) and mean platelet volume (C) were analyzed using EDTA-anticoagulated whole blood from hGPIbαFW and hGPIbαFW/cIL-4Rα mice on a Sysmex blood analyzer. Graphs show mean ± SEM of 20 and 13 mice, respectively; ∗∗∗∗P < .0001. (D-E) The effect of ILR4 expression on flnA distribution was evaluated in hGPIbαFW/cIL-4Rα bone marrow megakaryocytes using confocal microscopy. (D) Fluorescence intensity ratios of peripheral relative to the cytoplasmic flnA (5 random 1 μm2 regions from the central and peripheral regions of hGPIbαWT and hGPIbαFW megakaryocytes, as depicted in supplemental Figure 4); ∗∗∗∗P < .0001, ∗∗∗P < .001, 1-way ANOVA with Bonferroni post-test. At least 10 megakaryocytes were analyzed from each of 4 hGPIbαFW and 3 hGPIbαFW/cIL-4R mice. (E) Representative confocal images of hGPIbαFW and hGPIbαFW/cIL-4Rα bone marrow megakaryocytes showing homogenous flnA and organized CD41 distribution throughout the cytoplasm in hGPIbαFW/cIL-4Rα mice, in contrast to periphery flnA and disorganized CD41 in hGPIbαFW (93× glycerol objective; scale bar, 10 μm). (F-G) The DMS of hGPIbαFW/cIL-4Rα mice was analyzed after staining of femoral cryosections with anti-CD41 Ab and STED imaging. (F) Quantitation of the area of platelet demarcating territories within the cytoplasm of at least 5 megakaryocytes from each of 3 pairs of mice; ∗∗∗∗P < .0001 (G) Representative STED image demonstrating well-organized DMS in hGPIbαFW/cIL-4Rα bone marrow megakaryocytes, relative to hGPIbαFW cells (93× glycerol objective; scale bar, 10 μm). (H-K) Quantitation of bud diameter (performed as described under Methods, H), total CD41+ buds (I), interstitial buds (J) and MK location (intrasinusoidal), sinusoidal contact (SC), or bone marrow hematopoietic compartment (BMHC) (K) per 370 μm2 field generated from tile scans in hGPIbαFW and hGPIbαFW/cIL-4Rα. Graphs show the mean ± SEM of 3 mice; ∗P < .05, ∗∗∗∗P < .0001, unpaired 2-tailed t test or 2-way ANOVA as appropriate. Ab, antibody; ANOVA, analysis of variance; SEM, standard error of the mean.

Expression of cIL-4Rα ameliorates macrothrombocytopenia. (A) Anticoagulated whole blood from and hGPIbαFW and hGPIbαFW/cIL-4Rα mice was assessed for platelet GPIbα surface expression by flow cytometry using anti-CD42b Ab-PE. The results are the mean fluorescence intensity of gated platelet population (mean ± SEM of n = 10 pairs hGPIbαFW and hGPIbαFW/cIL-4Rα mice). (B-C) Platelet count (B) and mean platelet volume (C) were analyzed using EDTA-anticoagulated whole blood from hGPIbαFW and hGPIbαFW/cIL-4Rα mice on a Sysmex blood analyzer. Graphs show mean ± SEM of 20 and 13 mice, respectively; ∗∗∗∗P < .0001. (D-E) The effect of ILR4 expression on flnA distribution was evaluated in hGPIbαFW/cIL-4Rα bone marrow megakaryocytes using confocal microscopy. (D) Fluorescence intensity ratios of peripheral relative to the cytoplasmic flnA (5 random 1 μm2 regions from the central and peripheral regions of hGPIbαWT and hGPIbαFW megakaryocytes, as depicted in supplemental Figure 4); ∗∗∗∗P < .0001, ∗∗∗P < .001, 1-way ANOVA with Bonferroni post-test. At least 10 megakaryocytes were analyzed from each of 4 hGPIbαFW and 3 hGPIbαFW/cIL-4R mice. (E) Representative confocal images of hGPIbαFW and hGPIbαFW/cIL-4Rα bone marrow megakaryocytes showing homogenous flnA and organized CD41 distribution throughout the cytoplasm in hGPIbαFW/cIL-4Rα mice, in contrast to periphery flnA and disorganized CD41 in hGPIbαFW (93× glycerol objective; scale bar, 10 μm). (F-G) The DMS of hGPIbαFW/cIL-4Rα mice was analyzed after staining of femoral cryosections with anti-CD41 Ab and STED imaging. (F) Quantitation of the area of platelet demarcating territories within the cytoplasm of at least 5 megakaryocytes from each of 3 pairs of mice; ∗∗∗∗P < .0001 (G) Representative STED image demonstrating well-organized DMS in hGPIbαFW/cIL-4Rα bone marrow megakaryocytes, relative to hGPIbαFW cells (93× glycerol objective; scale bar, 10 μm). (H-K) Quantitation of bud diameter (performed as described under Methods, H), total CD41+ buds (I), interstitial buds (J) and MK location (intrasinusoidal), sinusoidal contact (SC), or bone marrow hematopoietic compartment (BMHC) (K) per 370 μm2 field generated from tile scans in hGPIbαFW and hGPIbαFW/cIL-4Rα. Graphs show the mean ± SEM of 3 mice; ∗P < .05, ∗∗∗∗P < .0001, unpaired 2-tailed t test or 2-way ANOVA as appropriate. Ab, antibody; ANOVA, analysis of variance; SEM, standard error of the mean.

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