Figure 8

VLZ stimulates megakaryocytopoiesis via the 5-HT1A receptor. (a) Representative immunoblot images and biochemical quantification of 5-HTR1A after treatment with VLZ (2.5, 5, and 10 μM) in Meg-01 cells for 5 days (n=3 per group). (b) The DARTS assay for target validation. 5-HTR1A protein stability was increased upon VLZ (200 μM) treatment in Meg-01 lysates. Pronase was added using several dilutions (1:500, 1:1000, or 1500) from 50 μg/mL stock for 10 min at 40 °C (n=3 per group). (c) The DARTS assay demonstrated the dose-dependent binding of VLZ to 5-HTR1A in Meg-01 cells. Treatment with pronase (1:1000) was conducted for 10 min at 40 °C (n=3 per group). (d) Immunofluorescence analysis of the expression of 5-HTR1A in Meg-01 cells after VLZ (2.5, 5, and 10 μM) intervention for 5 days. Cells were stained with DAPI for nuclei (blue) and antibodies for 5-HTR1A (green). Bars represent 100 μm. (e–k) Meg-01 cells were treated with VLZ (10 μM), WAY-100635 (2.5 μM), VLZ (10 μM)+WAY-100635 (2.5 μM) for 5 days. (e) Representative images, bars represent 25 μm. (f) Giemsa staining of Meg-01 cells, bars represent 100 μm. (g) Phalloidin staining of Meg-01 cells, bars represent 100 μm. (h, i) Flow cytometry analysis of the expression of CD41/CD42b and the DNA ploidy. (j, k) The histogram shows the percentage of CD41+/CD42b+ cells and DNA ploidy for each group. (l) Western blot analysis of 5-HTR1A, RAS and ERK expression after Meg-01 cells were treated with VLZ (10 μM), WAY-100635 (2.5 μM), and VLZ (10 μM)+WAY-100635 (2.5 μM) for 5 days. The histogram shows the expression of 5-HTR1A, RAS, and ERK in each group (n=3 per group). The data represent the mean ± SD of three independent experiments. *p≤0.05, **p≤0.01, and ***p≤0.001, ns: no significance, vs the control group.