Fig 2: Nogo deficiency attenuates obesity-induced systemic inflammation. The following assays were conducted on the serum and tissues collected from mice used in Figure 2. A, serum inflammatory cytokines were determined by the mouse cytokine array G3 (RayBiotech) method (n = 2). B, H&E staining was conducted on the eWAT sections, and the number of crown-like structure (CLS) was determined. C–E, immunohistochemical staining was conducted on the liver, BAT, and islet sections to determine MOMA2 expression with quantitative analysis of MOMA2-positive cells (n = 6). Fractions of M1 and M2 macrophages were determined on eWAT (F and G) and BAT (H and I) by FACS (n = 4). #p < 0.05, ##p < 0.01, ###p < 0.001 versus control HFD-fed mice. BAT, brown adipose tissue; eWAT, epididymal white adipose tissue; FACS, fluorescence-activated cell sorting; HFD, high-fat diet; NC, normal chow; Nogo, reticulon-4.

Fig 3: Nogo siRNA ameliorates HFD-induced bodyweight gain and associated metabolic disorders in mice. After 25 weeks of HFD feeding, the obese mice (C57BL/6J, male, ~31 weeks old) were divided into two groups (five mice per group) randomly and received tail vein injection of Nogo siRNA (Nogo KD) or scrambled siRNA (control) once every 3 days for five times. A, during the treatment, mouse bodyweight was determined at the indicated time points, and the average bodyweight change was calculated. At the end of experiment (3 days after the last siRNA injection), mouse body temperature was measured (B). After sacrifice, mouse tissues were collected for the following assays. Mouse liver, iBAT, and WAT were weighed and calculated as percent of tissue weight in bodyweight (C); TG levels in liver, serum, and skeletal muscle were determined by the assay kit or the automatic biochemical analyzer (D). Protein expression of Nogo-B, p-AMPKa, AMPKa, p-AKT, AKT2, and PGC-1a in liver or skeletal muscle (E and F), NF-?B p65 in liver, skeletal muscle, and WAT whole extract (G), liver nuclear or cytosolic extract (H) was determined by Western blot with quantitative analysis of band density; expression of Il-1ß, Tnf-a, Il-6, Arg-1, and iNOS mRNA in liver, skeletal muscle, and WAT (I) was determined by qRT–PCR. *p < 0.05, **p < 0.01, ***p < 0.001 versus control mice, n = 5. AMPKa, AMP-activated kinase a; Arg-1, arginase 1; HFD, high-fat diet; iBAT, interscapular brown adipose tissue; IL, interleukin; iNOS, inducible NO synthase; Nogo, reticulon-4; p-AMPKa, phosphorylated AMPKa; PGC-1a, peroxisome proliferator–activated receptor gamma coactivator 1a; qRT, quantitative RT; TG, triglyceride; Tnf, tumor necrosis factor; WAT, white adipose tissue.

Fig 4: Nogo deficiency enhances browning of adipose tissue in HFD feeding or cold exposure situation by promoting expression of thermogenic genes. At the end of experiment as indicated in Figure 2, iBAT was collected, and the tissue sections were conducted H&E staining with quantitation of adipocyte size (A, n = 6) and UCP-1 immunohistochemical staining (C) with quantitation of mean density (MD) (D, n = 5). Expression of thermogenic genes (Ucp-1, Tbx-1, and Cidea; B), TG hydrolysis–related genes (Hsl and Atgl; E), and fatty acid oxidation–related genes (Cpt-1ß, Cox5b, Cox8b, and Ndufb2; F) were determined by qRT–PCR (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 versus control NC-fed mice; #p < 0.05, ##p < 0.01, ###p < 0.001 versus control HFD-fed mice. Male littermate control and Nogo-/- mice (~10 weeks old, five mice per group) were housed at the 24 h cycles of 12 h at 4 °C and 12 h at room temperature for 7 days. At the end of experiment, bodyweight (G) and body temperature (H) were measured. iBAT, sc-WAT, and eWAT were collected, weighed, and calculated as percent of tissue weight in bodyweight (I). sc-WAT and iBAT sections were conducted H&E staining (J and L) and UCP-1 immunohistochemical staining with quantitation of MD (N and O). Expression of Ucp-1 in sc-WAT; Ucp-1, Prdm16, and Dio2 mRNA in iBAT were determined by qRT–PCR (n = 5; K and M). &p < 0.05, &&p < 0.01, &&&p < 0.001 versus control mice. Atgl, adipose TG lipase; Cidea, cell death–inducing DNA fragmentation factor-alpha–like effector a; Cox5b, cytochrome C oxidase subunit 5b; Cox8b, cytochrome C oxidase subunit 8b; Cpt-1ß, carnitine palmitoyltransferase 1ß; Dio2, deiodonase 2; eWAT, epididymal white adipose tissue; HFD, high-fat diet; Hsl, hormone-sensitive lipase; iBAT, interscapular brown adipose tissue; NC, normal chow; Ndufb2, NADH:ubiquinone oxidoreductase subunit b2; Nogo, reticulon-4; Prdm16, PR domain containing 16; qRT, quantitative RT; sc-WAT, subcutaneous white adipose tissue; Tbx-1, T-box transcription factor 1; TG, triglyceride; UCP-1, uncoupling protein-1.

Fig 5: Lack of Nogo expression promotes healthy expansion of adipose tissue by inhibiting fibrosis and collagen synthesis. The following assays were conducted for eWAT and sc-WAT tissues collected from mice used in Figure 2. eWAT sections were conducted H&E staining (A) with quantitation of adipocyte size (B, n = 6) and Sirius red staining (G, n = 5). C–F, expression of adiponectin, leptin, Vegf-ß, and Col6a2 mRNA in eWAT was determined by qRT–PCR (n = 6). sc-WAT sections of HFD-fed littermate control and Nogo-/- mice (it is hardly to collect sc-WAT when the animals were fed normal chow, Fig. 2H) were conducted H&E staining (H) with quantitation of adipocyte size (I, n = 6) and Sirius red staining (N, n = 5). J–M, expression of adiponectin, leptin, Vegf-ß, and Col6a1 mRNA in sc-WAT was determined by qRT–PCR (n = 6). #p < 0.05, ##p < 0.01, ###p < 0.001 versus control HFD-fed mice. Col6a1, collagen type VI a1; Col6a2, collagen type VI a2; eWAT, epididymal white adipose tissue; HFD, high-fat diet; NC, normal chow; Nogo, reticulon-4; qRT, quantitative RT; sc-WAT, subcutaneous white adipose tissue; Vegf-ß, vascular endothelial growth factor ß.