Hepatocyte Notch1 deletion impairs hepatic TPO production in vivo. Normal platelets were desialylated, and a total number of 2.5 × 10⁸ desialylated platelets was infused into Notch1^fl/fl and HC Notch1^−/− mice (A), followed by isolation of the liver protein (B) to measure the expression of TPO (C), HES5 (D), and phosphorylation of STAT3 (E) by western blot (mean ± SD; n = 3; 2-way ANOVA) or extraction of liver RNA to detect the TPO mRNA level 24 hours after infusion of desialylated platelets (fold change relative to the basal level) by quantitative PCR assay (mean ± SD; n = 3; unpaired student t test) (F). (G) Wild-type mice were IV injected with vehicle or neuraminidase (50 mU/mouse) to induce in vivo desialylation. After 2 hours, liver was isolated for immunostaining of platelets (CD41, red), liver sinusoidal endothelial cells (CD31, blue), or hepatocytes (autofluorescence, green). The images were analyzed by confocal microscopy (100× oil objective). To observe the colocalization of platelets with hepatocytes, 100× 3-dimensional z stacked image was reconstructed. Arrowhead indicates the colocalization of a platelet with hepatocytes. Platelets/hepatocytes colocalization (%) was quantified as the number of colocalized platelets relative to the total number of platelets in each observed field (100×) (mean ± SE; n = 5-6; unpaired student t test). (H) Untreated or desialylated platelets were fixed, dehydrated, dried, and coated with gold, followed by analysis of the structural changes under a scanning electron microscopy (20 000×) (Representative images were shown from 3 independent experiments). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.