Fig 2: Lower representation of CD56+/CD45+ cells in duodenum, ileum, and colon from GF mice, compared with Conv-R mice. A, B) Representative immunofluorescence images showing CD56+/CD45+ cells in the colon from Conv-R (A) and GF (B) mice. Scale bars, 50 µm. C) Total counts of cells expressing both CD56 and CD45 in Conv-R and GF mice. Conv-R, n = 5; GF, n = 5. Data represent means ± sd. *P < 0.05 (nonparametric Mann-Whitney test).

Fig 3: Experimental validation of NCAM1, PTPRD, and DCC as BACE1 substrates. Western blots were performed on protein lysates from BACE1 knockout (KO) and wildtype (WT) brain endothelial cells (BECs) or hCMEC/D3 treated with M-3 or transfected with BACE1. Relative protein expression of full length NCAM1 (A,D,G), PTPRD (B,E,H), and DCC (C,F,I) after normalisation to beta actin. PTPRD and DCC gene expression (J,K) in hCMEC/D3. N = 3 per group, * denotes p < 0.05.

Fig 4: The synergistic effect of Dll4 inhibition on CAL in fat grafts is associated with enhanced vascular integrity and relieved inflammation. (A) Heatmap of unsupervised clustering of normalized enrichment score (NES) of REACTOME pathways by single-sample gene set enrichment analysis (ssGSEA) among control, Dll4 inhibition, cell-assisted lipotransfer (CAL), and CAL plus Dll4 inhibition group samples (n = 4 each). (B) REACTOME pathways were significantly positively enriched among the CAL plus Dll4 inhibition group. Note that NCAM1 and GAP junction-related pathways were enriched after Dll4 inhibition. (C) Representative images and quantification of NCAM1 and Connexin43 in the control, CAL, and CAL plus Dll4 inhibition groups (n = 6). (D) Selected REACTOME pathways that were significantly negatively enriched among CAL plus Dll4 inhibition group. Note that inflammation related pathways including cytokine, interferon, and CD28-related pathways were suppressed after Dll4 inhibition. P-values were calculated comparing CAL samples and CAL plus Dll4 inhibition samples. (E) Representative images and quantification of FOXP3+CD3+ and CD206+F4/80+ cells in the control, CAL, and CAL plus Dll4 inhibition groups (n = 6). Values are presented as the mean ± standard deviation. **P < .01, ***P < .001 vs control group. ### P < .001 vs the CAL group. Scale bars = 100 µm.

Fig 5: BACE1 proteolysis of SEZ6, APP, and CHL1 in the hippocampus and olfactory bulb with age.A, representative immunoblot of PBS soluble (Soluble) and membrane (Membrane) fractions of the hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1-/- mice. Well-characterized BACE1 substrates are also analyzed in the hippocampus and olfactory bulb at three different ages using anti-SEZ6 (14E5; kindly provided by Dr S. Lichtenthaler), anti-CHL1 (AF2147), anti-sAPPß (BAWT; kindly provided by Dr S. Lichtenthaler), anti-APP (C1/6.1), anti-NCAM1 (AF-2408), anti-NCAM2 (sc-136328), anti-BACE1 (D10E5), anti-GAPDH (MAB374), and anti-calnexin (610523) antibodies. The molecular weight of sSEZ6ß in HC (~155 kDa, arrowhead) is different from the one in the OB (~165 kDa, double arrowheads) at the age of 4 and 12 months. The weak band at ~140 kDa (asterisk) represents immature SEZ6-FL, while the strong band at 165-kDa (open arrowhead) or 175-kDa (double open arrowheads) in the OB represents mature SEZ6-FL. The different molecular weight of sSEZ6ß or SEZ6-FL between the HC and OB of 4- and 12-month-old mice is most likely due to alternative splicing or posttranslational modifications. In the HC membrane fraction, CHL1-FL was detected at ~185 kDa at P10, while CHL1-FL is detected as two bands at ~185 kDa (plain arrow) and ~175 kDa (dash arrow) in 4- and 12-month-old mice, most likely due to splicing variant or glycosylation. B–G, graphs represent densitometry of soluble protein (sSEZ6ß, sAPPß, and sCHL1) normalized to GAPDH or densitometry of membrane protein (mature SEZ6-FL, APP-FL, and 185-kDa CHL1) normalized to calnexin. White and gray bars represent BACE1+/+ and BACE1-/- mice, respectively. Unpaired two tailed t-test was used for analysis. *p < 0.05, **<0.01, ***p < 0.001, ****p < 0.0001, ns; not significant, P10 (BACE1+/+; n = 4–5, BACE1-/-; n = 4–5), 4 months (BACE1+/+; n = 5–6, BACE1-/-; n = 5), 12 months (BACE1+/+; n = 4–5, BACE1-/-; n = 4–5).