Nucleophosmin3 carried by small extracellular vesicles contribute to white adipose tissue browning

Figures

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  Fig. 1. NPM3 was enriched in BAT and decreased in obesity. A IHC analysis of NPM3 expression in different tissues (WAT, BAT, Liver and Muscle) from 8-week-old male mice, the boxed region was shown at higher magnification at right (scale bar = 50 μm), the expression of NPM3 was pointed by red arrows; B qRT-PCR analysis of relative NPM3 mRNA expressions in ASCs and adipocytes from inguinal adipose tissue (iWAT), epididymal adipose tissue (eWAT) and BAT, data were represented as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s test, *P < 0.05, **P < 0.01; C AAV-NPM3-EGFP infected BAT was transplanted into the inguinal site of the 8-week old mice and the expressions of EGFP were detected in the iWAT, muscle, liver, lung and kidney after 1 week; D Relationship of plasma NPM3 levels of mice across different bodyweight groups (n = 13) (Linear regression analysis); NPM3 protein expression (n = 3) in iWAT (E), eWAT (F) and BAT (G) of 3-month-old male ob/ob mice and lean mice, data were represented as mean ± SEM and analyzed by student t test, *p < 0.05

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  Fig. 2. NPM3 promoted WAT browning by stabilizing PRDM16 mRNA. Relative mRNA expressions of PGC-1α, CIDEA and UCP1 after browning induction in 3T3-L1 preadipocytes (3T3-L1 diff) transfected with or without NPM3 overexpression plasmids (NPM3-OE diff) (A) or siRNAs (siNPM3 diff) (B) (n = 3), data were represented as mean ± SEM and analyzed by student t test; *P < 0.05, **P < 0.01, ***P < 0.001; C Glucose consumption in 3T3-L1 preadipocytes after transfected with NPM3 overexpressed plasmids or siNPM3 (n = 3), data were represented as mean ± SEM and analyzed by student t test, *P < 0.05, **P < 0.01; D Relative mRNA expressions of PGC-1α, CIDEA and UCP1 in iWAT with or without CL-316,243/siNPM3 treatment (n = 5), data were represented as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s test, *P < 0.05, **P < 0.01, ***P < 0.001; E Representative HE and UCP1 immunohistochemical staining in iWAT with or without CL-316,243/siNPM3 treatment, scale bar = 100 μm, the boxed region was shown at higher magnification at right (scale bar = 50 μm); F Western blot to confirm immunoprecipitation (IP) of NPM3; 10% IP cell lysate was used as the input; G Enrichment of selected targets was compared…

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  Fig. 3. NPM3 was exclusively localized to sEVs. A Cell immunofluorescence assay confirming the presence of NPM3-EGFP in recipient cells via the transwell assay, GW4869 or Calpeptin was used to block sEVs release, scale bar = 50 μm; B Differential centrifugation procedure for the isolation of sEVs from the supernatants of digested adipose tissue (SN-AT); C The size of different types of EVs (lEVs, mEVs, sEVs) were detected using dynamic light scattering; D NPM3 expressions in different sizes of EVs and different centrifugations supernatants; E Comparison of NPM3 and sEVs marker proteins in 14 fractions (F1–F14) isolated from sucrose density-gradient centrifugation; F Comparison of NPM3, CD63, IGFBP3 in sEVs treated with proteinase K and/or Triton-X-100

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  Fig. 4. Knocking down NPM3 in BAT impaired sEVs mediated WAT browning. A Relative mRNA expressions of PGC-1α, CIDEA and UCP1 in 3T3-L1 preadipocytes treated with sEVs isolated by TEI or ultracentrifugation method (n = 3), data were represented as mean ± SEM and analyzed by student t test, *P < 0.05,, **P < 0.01; B A flow chart depicting the isolation of sEVs-BAT and sEVs-BAT-siNPM3; C qRT-PCR analysis of thermogenic related mRNA, miRNAs in the sEVs-BAT and sEVs-BAT-siNPM3 (n = 3); D Relative mRNA expressions of PGC-1α, CIDEA and UCP1 in 3T3-L1 preadipocytes treated with sEVs-BAT or sEVs-BAT-siNPM3 (n = 3), data were represented as mean ± SEM and analyzed by student t test, *P < 0.05, **P < 0.01; E Glucose consumption ratio of 3T3-L1 preadipocytes after treated with sEVs-BAT and sEVs-BAT-siNPM3 (n = 3), data were represented as mean ± SEM and analyzed by student t test, *P < 0.05; F Representative UCP1 immunohistochemical staining in iWAT and eWAT in mice treated with sEVs-BAT or sEVs-BAT-siNPM3, Scale bar = 100 μm; G Relative mRNA expressions of PGC-1α, CIDEA and UCP1 in iWAT and eWAT in mice treated with sEVs-BAT or sEVs-BAT-siNPM3 (n = 6), data were represented as mean ± SEM and…

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  Fig. 5. Knocking down NPM3 in BAT blunted sEVs mediated insulin sensitivity in HFD-fed mice. A Schematic study plan where 8 weeks old, male C57BL/6 mice were fed with chow or HFD for 15 weeks, HFD-fed mice were injected in vein with sEVs-BAT and sEVs-BAT-siNPM3 during the last 9 weeks; HFD-fed mice treated with PBS were used as controls; B Body weight change during the experiment (n = 6) was evaluated after the intervention, data were represented as meanSEM and analyzed by one-way ANOVA followed by Tukey’s test, **P < 0.01; C Diet intake (Kcal/group); D iWAT, eWAT weight was evaluated after of intervention, data were represented as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s test, **P < 0.01, ***P < 0.001; E H&E staining of iWAT and eWAT were evaluated after of intervention, the size of adipocytes in eWAT of HFD fed mice was evaluated after the intervention (5 images per group), data were represented as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s test, **P < 0.01, ***P < 0.001; F Fasting glucose levels (fasting duration 6 h) were evaluated after the intervention, data were represented as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s test,…