Figure 4.
YAP1 promotes thrombopoiesis through F-actin reorganization. (A) Proplatelet formation in MKs from patients with ITP and healthy controls after immunostaining for F-actin (yellow, phalloidin) and MKs (red, CD41). The arrows indicate proplatelet formation. Scale bar, 10 μm. Right, assessment of proplatelet formation in MKs from patients with ITP and healthy controls (n = 6). (B) MKs were isolated from WT and YAP1+/− mice to observe the cytoskeleton and proplatelet formation. Right: proplatelet numbers were calculated for WT and YAP1+/− mice (n = 10). (C) Confocal images of MKs from the YAP1 upregulation or reduction group after immunostaining for actin/stress fibers (yellow, phalloidin) and MKs (red, CD41). Scale bar, 10 μm. (D) The number of proplatelet formation (top) was analyzed and percentage of proplatelet formation (bottom) were assessed by flow cytometry (n ≥ 10). (E) Flow cytometry analysis of the percentage of CD41+ MKs and CD41+CD42+ MKs after XMU-MP-1 and blebbistatin treatment in D7, D10, and D12 (n = 3). (F-G) Measurement for adhered (F) and migrated (G) MKs in the untreated group and YAP1 activation (XMU-MP-1) or inhibition (Peptide 17) groups after blebbistatin (5 μM), cytochalasin D (500 nM), and narciclasine (50 nM) treatment (n ≥ 3). (H-J) Confocal images of MK thrombopoiesis in the untreated group (H) and YAP1 activation (XMU-MP-1; I) or inhibition (Peptide 17; J) groups after blebbistatin, cytochalasin D, and narciclasine treatment, after immunostaining for F-actin/stress fibers (yellow, phalloidin) and MKs (red, CD41). Scale bar, 10 μm. (K-L) Measurement of proplatelet formation in YAP1 activation (XMU-MP-1; K) or inhibition (Peptide 17; L) groups after blebbistatin, cytochalasin D, and narciclasine treatment (n ≥ 5). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. B, blebbistatin; XB, XMU-MP-1 + blebbistatin; Cy, cytochalasin D; N, narciclasine.

YAP1 promotes thrombopoiesis through F-actin reorganization. (A) Proplatelet formation in MKs from patients with ITP and healthy controls after immunostaining for F-actin (yellow, phalloidin) and MKs (red, CD41). The arrows indicate proplatelet formation. Scale bar, 10 μm. Right, assessment of proplatelet formation in MKs from patients with ITP and healthy controls (n = 6). (B) MKs were isolated from WT and YAP1+/− mice to observe the cytoskeleton and proplatelet formation. Right: proplatelet numbers were calculated for WT and YAP1+/− mice (n = 10). (C) Confocal images of MKs from the YAP1 upregulation or reduction group after immunostaining for actin/stress fibers (yellow, phalloidin) and MKs (red, CD41). Scale bar, 10 μm. (D) The number of proplatelet formation (top) was analyzed and percentage of proplatelet formation (bottom) were assessed by flow cytometry (n ≥ 10). (E) Flow cytometry analysis of the percentage of CD41+ MKs and CD41+CD42+ MKs after XMU-MP-1 and blebbistatin treatment in D7, D10, and D12 (n = 3). (F-G) Measurement for adhered (F) and migrated (G) MKs in the untreated group and YAP1 activation (XMU-MP-1) or inhibition (Peptide 17) groups after blebbistatin (5 μM), cytochalasin D (500 nM), and narciclasine (50 nM) treatment (n ≥ 3). (H-J) Confocal images of MK thrombopoiesis in the untreated group (H) and YAP1 activation (XMU-MP-1; I) or inhibition (Peptide 17; J) groups after blebbistatin, cytochalasin D, and narciclasine treatment, after immunostaining for F-actin/stress fibers (yellow, phalloidin) and MKs (red, CD41). Scale bar, 10 μm. (K-L) Measurement of proplatelet formation in YAP1 activation (XMU-MP-1; K) or inhibition (Peptide 17; L) groups after blebbistatin, cytochalasin D, and narciclasine treatment (n ≥ 5). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. B, blebbistatin; XB, XMU-MP-1 + blebbistatin; Cy, cytochalasin D; N, narciclasine.

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