Fig. 2: The decreased expression of METTL3 is related to the poor prognosis of patients with metabolic memory damage in diabetes.

A mRNA isolated from patients with diabetes wounds and paired normal skin were used in Dot blot analysis with an anti-m⁶A antibody, and MB (methylene blue) staining as load control (Representative image in the left panel). Calculate the relative content of messenger ribonucleic acid m6A in diabetes wound tissue and paired normal skin tissue (Right panel) (n = 5 per group). B The mRNA isolated from HUVECs of different treatment groups was subjected to dot blot analysis using the m⁶A antibody (upper panel). MB staining serves as a loading control and the results were quantified and processed (right panel) (n = 3 per group). C The heatmap of cluster analysis based on transcriptome RNA-Seq of MM and Nr-T. D Venn diagram of differential genes and m6A-related genes in vascular endothelial cells of different treatment groups compared to control cells. E METTL3 protein levels were measured in HUVECs of different treatment groups by western blotting and the results were quantified and processed (top panel). and Nr-CWS treatment group by western blotting and the results were quantified and processed (down panel) (n = 3 per group). F The protein levels of METTL3 in HUVECs with METTL3 overexpression were measured by western blotting (n = 3 per group). G Tube formation assay and cell growth in HUVECs with METTL3 overexpression or from the corresponding control cells. Tubes were imaged (scale bars = 100 µm, left panel), and tube formation and cell growth were quantified (right panel) (n = 3 per group). H EDU staining of HUVECs with Mettl3 overexpression or from the corresponding control cells. Positive cells were imaged (scale bars = 100 µm, left panel) and quantified (right panel) (n = 3 per group). I The protein levels of METTL3 in HUVECs with wild-type or catalytic mutant METTL3 overexpression were measured by western blotting (left panel). The mRNAs isolated from wild-type or catalytic mutant Mettl3-overexpressing HUVECs were used in dot blot analyses with m6A antibody (right panel). MB staining served as a loading control (n = 3 per group). J The tube formation assay in HUVECs with wild-type or catalytic mutant Mettl3 overexpression was determined (left panel). The tubes were imaged (scale bar = 100 µm, left panel) and tube formation was quantified (right panel) (n = 3 per group). K Mettl3 expression was knocked down by siRNA in HUVECs and measured by western blotting (upper panel). The mRNAs isolated from si-Mettl3 or si-Control HUVECs were used in dot blot analyses with m6A antibody (bottom panel). MB staining served as a loading control. L The tube formation assay of Mettl3 knocked down by siRNA or si-Control in HUVECs. Tubes were imaged (scale bars = 100 µm, left panel), and tube formation was quantified (right panel) (n = 3 per group). M EDU staining of HUVECs with Mettl3 knocked down by siRNA or si-Control. Positive cells were imaged (scale bars = 100 µm, left panel) and quantified (right panel) (n = 3 per group). N Total cellular ATP levels were measured in HUVECs with Mettl3 knocked down by siRNA or si-Control starved for the indicated times (n = 3 per group). O Cellular ROS levels in si-control and Mettl3 knockdown HUVECs (left panel). The ROS was quantified (right panel) (n = 3 per group). P Representative wound images at d 0 and 14 during the healing process in animals treated with sense or AS-ODN. And KRT14 immunohistochemical staining of 14-day wound tissue. Wound diameters were measured on indicated days post-wounding from days 0–14 (the number of injections is 2) (n = 4 per group). Q METTL3 protein in these wounds was analyzed by western blotting, and relative METTL3 expression was normalized to β-actin using ImageJ densitometry (n = 4 per group). R Wound sections (day 14) were stained for CD31 (red) and α-SMA (green) to visualize stratified epithelial cells and counterstained with DAPI (scale bars = 50 µm). S A laser Doppler imager show the blood flow perfusion in the wounds that were subjected to different treatments (upper panel) and Quantification of wound perfusion in the wounds (down panel) (n = 4 per group). The data are represented as mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 are considered significant. α-SMA α-smooth muscle actin, HUVECs human umbilical vein endothelial cell.