Fig. 1
Generation of megakaryocytic lineage-specific Becn1 knockout Becn1f/f;Pf4-iCre mice. a–c Generation of Becn1f/f;Pf4-iCre mice, identified by electrophoresis of tail DNA and analysis of Becn1 mRNA expression levels by RT‒qPCR in CD41+ cells and CD41– cells from the BM. Gapdh was used as a reference gene. d Analysis of Becn1 expression (green) in MKs from Becn1f/f and Becn1f/f;Pf4-iCre mice by imaging flow cytometry. MKs were identified by CD41 staining. The upper panel shows a representative flow image, and the lower panel shows the results of the flow image analysis. e Analysis of the Becn1 protein expression level in platelets by western blotting. β-Actin was used as a loading control. f Analysis of Becn1 expression (green) in platelets from Becn1f/f and Becn1f/f;Pf4-iCre mice by confocal microscopy. Platelets were labeled with CD41 (red). g, h Western blot analysis of Becn1 expression in whole BM cells, CD41–cells from the BM, and MNCs from the peripheral blood, kidney, spleen, liver, and lung. i Volcano plot of the platelet proteome. Differentially expressed proteins (DEPs) were determined under the conditions of |log2(fold change)| ≥ log2(1.5) and P < 0.05. j Heatmap of DEPs. k Gene set enrichment analysis (GSEA) of proteins involved in autophagy. The signaling pathway was adapted from the Gene Ontology (GO) pathway (GO:0006914). l GO enrichment analysis of DEPs in platelets. The ten most significant biological process (BP) terms. m Western blot analysis of autophagy-related proteins in platelets with or without starvation. n Colocalization analysis of LC3 and Lamp1 in platelets using confocal microscopy. Data are means ± SEMs. *P < 0.05; **P < 0.01
