Fig. 2.

The impaired haematopoiesis-supporting ability of HUVECs was restored by activating autophagy via Beclin-1 upregulation. (a) Beclin-1 mRNA levels were analysed by qRT-PCR after Beclin-1 knockdown (H-sBECN) and overexpression (H-sB/pB) in HUVECs. The intracellular levels of (b) Beclin-1, (c) LC3 and (d) p62 in the H-sBECN, H-sB/pB and control groups were analysed by flow cytometry. (e) Representative western blots of LC3, Beclin-1 and p62. (f) Representative images (left panel) and quantification (right panel) of intracellular autophagosomes and autophagic vacuoles in the H-sBECN, H-sB/pB and control groups (original magnification, 10 × , scale bars represent 50 μm). The numbers of intracellular autophagosomes and autophagic vacuoles (green) per cell were determined in three random high-power fields and averaged. (g) Transmission electron microscope image of HUVECs in the H-sBECN, H-sB/pB and control groups. Double-membrane autophagosomes (open arrows) and single-membrane autolysosomes (filled arrows) containing degraded material clustered at perinuclear sites (magnification: above 6000 × , scale bars represent 2 μm; below 20,500 × , scale bars represent 200 nm). (h) The CFU plating efficiency and (i) apoptosis rate of BM CD34⁺ cells from healthy donors were analysed after 7 days of coculture with HUVECs. (j) HUVEC count and (k) representative images (left panel) and quantification (right panel) of the transwell migration assays for the H-sBECN, H-sB/pB and control groups (original magnification, 10 × , scale bars represent 50 μm). The numbers of migrated HUVECs per field of view were compared among the H-sBECN, H-sB/pB and control groups. (l) Intercellular ROS levels (MFI, mean±SEM) and (m) the apoptosis rate were analysed by flow cytometry. Wilcoxon's test for paired data was used to identify drug effects. All P-values <0.05 were considered significant and are provided in the figure. *P<0.05, **P<0.005, ***P<0.0005.