Figure 5.
Decreased proplatelet tip size and impaired microtubule organization of RhoB−/− MKs in vitro. (A) Quantification of MK numbers in wt and RhoB−/− cryosections of the fluorescently labeled BM. At least 10 fluorescence images/mouse were analyzed using a confocal microscope. Each data point represents 1 MK (n = 7). Unpaired, 2-tailed t test, mean plus or minus SD. (B) Representative images of ultrastructural analysis of wt and RhoB−/− MKs in situ using TEM. Scale bar, 4 µm. (C) Representative confocal microscopy images of femur cryosections. The femora were processed using the Kawamoto method for cryosections. Sections were stained with fluorescently labeled anti-CD105 antibody to stain vessels (red), and fluorescently labeled anti-GPIX antibody to visualize MKs (yellow) and DAPI (blue). Scale bar in overview, 40 µm; in zoom, 5 µm. (D) Quantification of MK localization in wt and RhoB−/− BM in images shown in panel B. (E-H) Analysis of proplatelet formation of BM-derived RhoB−/− MKs. (E) Quantification of in vitro proplatelet formation at 18 hours, 24 hours, and 48 hours after enrichment by bovine serum albumin (BSA) gradient. Graph shows percentage of MKs that perform PPF (n = 3 mice per genotype). Multiple unpaired 2-tailed Student t test with Holm-Sidak correction for multiple comparisons, mean plus or minus SD. (F) Quantification of proplatelet tip diameter of wt and RhoB−/− MKs 24 hours after BSA gradient (normalized to wt, n = 2 mice per genotype). At least 11 MKs per mice were analyzed. Each data point represents 1 measured proplatelet tip (wt = 250; RhoB−/− = 494). ***P < .001; Mann-Whitney U test, mean plus or minus SD. (G) Quantification of MT organization in enriched wt and RhoB−/− MKs (n = 3 mice per genotype). At least 13 images/mouse were analyzed. Mann-Whitney U test, mean plus or minus SD. (H) Representative confocal images of in vitro–differentiated MKs 24 hours after BSA gradient. The proplatelets were spun down to Poly-L-Lysine–coated glass slides and stained with phalloidin-Atto647 (magenta), α-tubulin-Alexa488 (cyan), and DAPI to visualize the nucleus. (n = 5) Scale bar in overview, 40 µm; inset, 5 µm. DAPI, 4′,6-diamidino-2-phenylindole; PPF, proplatelet-forming; Intrasin., intrasinusoidal; SC, sinusoidal contact; TB, tubulin.

Decreased proplatelet tip size and impaired microtubule organization of RhoB−/− MKs in vitro. (A) Quantification of MK numbers in wt and RhoB−/− cryosections of the fluorescently labeled BM. At least 10 fluorescence images/mouse were analyzed using a confocal microscope. Each data point represents 1 MK (n = 7). Unpaired, 2-tailed t test, mean plus or minus SD. (B) Representative images of ultrastructural analysis of wt and RhoB−/− MKs in situ using TEM. Scale bar, 4 µm. (C) Representative confocal microscopy images of femur cryosections. The femora were processed using the Kawamoto method for cryosections. Sections were stained with fluorescently labeled anti-CD105 antibody to stain vessels (red), and fluorescently labeled anti-GPIX antibody to visualize MKs (yellow) and DAPI (blue). Scale bar in overview, 40 µm; in zoom, 5 µm. (D) Quantification of MK localization in wt and RhoB−/− BM in images shown in panel B. (E-H) Analysis of proplatelet formation of BM-derived RhoB−/− MKs. (E) Quantification of in vitro proplatelet formation at 18 hours, 24 hours, and 48 hours after enrichment by bovine serum albumin (BSA) gradient. Graph shows percentage of MKs that perform PPF (n = 3 mice per genotype). Multiple unpaired 2-tailed Student t test with Holm-Sidak correction for multiple comparisons, mean plus or minus SD. (F) Quantification of proplatelet tip diameter of wt and RhoB−/− MKs 24 hours after BSA gradient (normalized to wt, n = 2 mice per genotype). At least 11 MKs per mice were analyzed. Each data point represents 1 measured proplatelet tip (wt = 250; RhoB−/− = 494). ***P < .001; Mann-Whitney U test, mean plus or minus SD. (G) Quantification of MT organization in enriched wt and RhoB−/− MKs (n = 3 mice per genotype). At least 13 images/mouse were analyzed. Mann-Whitney U test, mean plus or minus SD. (H) Representative confocal images of in vitro–differentiated MKs 24 hours after BSA gradient. The proplatelets were spun down to Poly-L-Lysine–coated glass slides and stained with phalloidin-Atto647 (magenta), α-tubulin-Alexa488 (cyan), and DAPI to visualize the nucleus. (n = 5) Scale bar in overview, 40 µm; inset, 5 µm. DAPI, 4′,6-diamidino-2-phenylindole; PPF, proplatelet-forming; Intrasin., intrasinusoidal; SC, sinusoidal contact; TB, tubulin.

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