Figure 5
Figure 5. Proplaletet formation by Tmod3−/− MKs in culture is impaired, and Tmod3−/− proplatelets have abnormal levels of VWF+ granules. (A) Immunofluorescence staining of α-tubulin in proplatelets produced by MKs cultured in vitro from Tmod3+/+ and Tmod3−/− fetal livers reveals uniformly small sizes for Tmod3+/+, but highly variable sizes for Tmod3−/− proplatelets, including some very large ones. Arrowhead and arrow (lower panel) are examples of a large and small Tmod3−/− proplatelet, respectively. Bar, 10 µm. (B) Percentages of MKs cultured in vitro that form proplatelets. ***P < .001. (C) Sizes of proplatelet buds produced by cultured MKs. Tmod3+/+, 5.23 ± 2.39 µm2 (n = 96); Tmod3−/−, 15.48 ± 9.31 µm2 (n = 98). ***P < .001. Proplatelet sizes (areas, µm2) were measured from α-tubulin–stained proplatelets using Volocity 6.3 software to circle their perimeter. (D) Immunofluorescence staining of VWF (green) and α-tubulin (red) in proplatelet buds from cultured MKs. Bar, 5 µm. (E) Total VWF per proplatelet based on the ratio of VWF fluorescence intensity vs α-tubulin intensity. Tmod3+/+, 0.80 ± 0.49 (n = 46); Tmod3−/−, 0.45 ± 0.27 (n = 48). ***P < .001. (F-G) Number of VWF+-stained puncta per proplatelet bud with respect to area (solid grey line for Tmod3+/+, solid black line for Tmod3−/−). The number of VWF+ granules per proplatelet bud was linearly dependent on proplatelet area for Tmod3+/+ but not Tmod3−/− MKs (R2 = 0.6017 vs R2 = 0.2112, respectively). However, the subset of Tmod3−/− proplatelet buds with areas smaller than or equal to the area of the largest Tmod3+/+ proplatelet bud (demarcated by blue dotted line), revealed a stronger linear correlation (R2 = 0.4945, dashed black line). Thus, in Tmod3−/− proplatelets, the overall weakness of the linear relationship of the number of VWF+ granules per proplatelet bud as a function of proplatelet area is driven primarily by the unusually large, aberrant proplatelet buds. Images are single optical sections acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a ×100 oil-immersion objective (N.A. 1.4) at zoom 0.6 (A) or zoom 2 (D).

Proplaletet formation by Tmod3−/− MKs in culture is impaired, and Tmod3−/− proplatelets have abnormal levels of VWF+ granules. (A) Immunofluorescence staining of α-tubulin in proplatelets produced by MKs cultured in vitro from Tmod3+/+ and Tmod3−/− fetal livers reveals uniformly small sizes for Tmod3+/+, but highly variable sizes for Tmod3−/− proplatelets, including some very large ones. Arrowhead and arrow (lower panel) are examples of a large and small Tmod3−/− proplatelet, respectively. Bar, 10 µm. (B) Percentages of MKs cultured in vitro that form proplatelets. ***P < .001. (C) Sizes of proplatelet buds produced by cultured MKs. Tmod3+/+, 5.23 ± 2.39 µm2 (n = 96); Tmod3−/−, 15.48 ± 9.31 µm2 (n = 98). ***P < .001. Proplatelet sizes (areas, µm2) were measured from α-tubulin–stained proplatelets using Volocity 6.3 software to circle their perimeter. (D) Immunofluorescence staining of VWF (green) and α-tubulin (red) in proplatelet buds from cultured MKs. Bar, 5 µm. (E) Total VWF per proplatelet based on the ratio of VWF fluorescence intensity vs α-tubulin intensity. Tmod3+/+, 0.80 ± 0.49 (n = 46); Tmod3−/−, 0.45 ± 0.27 (n = 48). ***P < .001. (F-G) Number of VWF+-stained puncta per proplatelet bud with respect to area (solid grey line for Tmod3+/+, solid black line for Tmod3−/−). The number of VWF+ granules per proplatelet bud was linearly dependent on proplatelet area for Tmod3+/+ but not Tmod3−/− MKs (R2 = 0.6017 vs R2 = 0.2112, respectively). However, the subset of Tmod3−/− proplatelet buds with areas smaller than or equal to the area of the largest Tmod3+/+ proplatelet bud (demarcated by blue dotted line), revealed a stronger linear correlation (R2 = 0.4945, dashed black line). Thus, in Tmod3−/− proplatelets, the overall weakness of the linear relationship of the number of VWF+ granules per proplatelet bud as a function of proplatelet area is driven primarily by the unusually large, aberrant proplatelet buds. Images are single optical sections acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a ×100 oil-immersion objective (N.A. 1.4) at zoom 0.6 (A) or zoom 2 (D).

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