Journal: Molecular Metabolism

Article Title: Deubiquitinating enzyme USP2 regulates brown adipose tissue thermogenesis via controlling EBF2 stabilization

doi: 10.1016/j.molmet.2025.102139

Figure Lengend Snippet: Overexpression of Usp2 in BAT ameliorates HFD-induced obesity and metabolic dysfunction. A. Schematic diagram of the in-situ injection of AAV-OeUsp2 into BAT for the establishment of the Usp2 BOE model, followed by HFD feeding. B. Body weight of Usp2 BOG and control mice housed at RT (22 °C). Usp2 BOE vs control, ( n = 5 vs 6). C. Lean mass and fat mass. Usp2 BOE vs control, ( n = 7 vs 9). D. The weight of indicated tissues ( n = 5). E. Rectal temperature of Usp2 BOE and control mice during acute cold exposure ( n = 8). F. Representative infrared images of Usp2 BOE and control mice following 4 h of acute cold exposure. G. Representative H&E staining of adipose tissue and liver in Usp2 BOE and control mice (Scale bar, 100 μm). H. Quantitative PCR analysis of thermogenic genes in BAT of Usp2 BOE and control mice. Usp2 BOE vs control, ( n = 4 vs 5). I. Representative Ucp1 immunostaining of BAT in Usp2 BOE and control mice (Scale bar, 100 μm). J-L. Oxygen consumption rate ( J ), carbon dioxide production rate ( K ) and heat production rate ( L ) of Usp2 BOE and control mice over 24-hour period monitored at RT (22 °C) ( n = 4). M-O. Average oxygen consumption rate ( M ), average carbon dioxide production rate ( N ) and average heat production rate ( O ) at day time or night time during the 24-hour of monitoring as in (J–L). P. Insulin tolerance test (ITT) results of mice after 8 weeks of HFD feeding ( n = 7). Q. Analysis of ITT results in P. R. Glucose tolerance test (GTT) results of mice after 8 weeks of HFD feeding ( n = 6). S. Analysis of GTT results in R. For statistical analysis, two-way ANOVA was performed in B, E, J-L, P and R. Unpaired, two-tailed t -tests were performed in C, D, H, M−O, Q and S. (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001). Each point represents a biological replicate. Data were presented as the mean ± S.E.M.

Article Snippet: Adipocyte-specific adeno-associated virus 2/9 (AAV Serotype 2/9, adiponectin promoter)-mediated overexpression of FLAG-tagged mouse Usp2, control (GFP), shRNA targeting Usp2, and scrambled control (NC) were constructed, amplified, and purified by OBiO Technology (Shanghai, China).

Techniques: Over Expression, In Situ, Injection, Control, Staining, Real-time Polymerase Chain Reaction, Immunostaining, Two Tailed Test

Journal: Molecular Metabolism

Article Title: Deubiquitinating enzyme USP2 regulates brown adipose tissue thermogenesis via controlling EBF2 stabilization

doi: 10.1016/j.molmet.2025.102139

Figure Lengend Snippet: EBF2 overexpression attenuates USP2 deficiency-potentiated thermogenic dysfunction. A. Schematic illustration of the establishment of three groups: AAV-NC + Adv-GFP vs AAV-shUsp2 + Adv-GFP vs AAV-shUsp2 + Adv-EBF2, n = 6 vs 5 vs 5. Image created with BioRender.com . B. Tissue weight of BAT. C. Tissue weight of iWAT and eWAT. D. Representative H&E staining of BAT. E. Representative UCP1 immunostaining of BAT. F. Immunoblot analysis of UCP1. G. Quantitative PCR analysis of thermogenic genes of BAT ( n = 5 vs 5 vs 5). H-J. Oxygen consumption rate ( H ), carbon dioxide production rate ( I ) and heat production rate ( J ) of mice over 24-hour period monitored at RT (22 °C) ( n = 4 vs 4 vs 4). K-M. Average oxygen consumption rate ( K ), average carbon dioxide production rate ( L ) and average heat production rate ( M ) at day time or night time during the 24-hour of monitoring as in (H–J). For statistical analysis, two-way ANOVA was performed in H-J. Unpaired, two-tailed t -tests were performed in B, C, G and K-M. (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001). Each point represents a biological replicate. Data were presented as the mean ± S.E.M.

Article Snippet: Adipocyte-specific adeno-associated virus 2/9 (AAV Serotype 2/9, adiponectin promoter)-mediated overexpression of FLAG-tagged mouse Usp2, control (GFP), shRNA targeting Usp2, and scrambled control (NC) were constructed, amplified, and purified by OBiO Technology (Shanghai, China).

Techniques: Over Expression, Staining, Immunostaining, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Adenosine generated through ATP hydrolysis exerts its wakefulness‐promoting effects by binding to A 1 receptors. A) Setup for local drug delivery through bilateral canula on top of PZ in C57BL/6J mice (top). Representative image of canula location (bottom). Scale bar, 100 µm. B) Hourly percentage of time spent in wakefulness, REM sleep and NREM sleep in 5 h after the administration of NECA ( n = 6 mice) or vehicle ( n = 6 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. Wake: * p = 0.0496, **** p < 0.0001; REM: * p = 0.0433; NREM: **** p < 0.0001. C) Relative EEG power (0‐30 Hz) during wakefulness after the administration of NECA ( n = 4 mice) or vehicle ( n = 4 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. n.s., p = 0.4756. D) Representative image of A1 receptor expression in the PZ by immunohistochemistry staining of A 1 receptors (red) and DAPI (blue) and a zoom‐in graph (right). Scale bar, 100 µm. E) Setup for optogenetic activation of astrocytes in the PZ following the administration of CPT (A 1 receptor inhibitor). F) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of CPT or vehicle. From NREM to: vehicle group ( n = 8 rats), CPT group ( n = 5 rats); From REM or Wake to: vehicle group ( n = 6 rats), CPT group ( n = 4 rats). Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001. G) Setup for simultaneous bilateral fiber photometry recordings of ATP and adenosine in the PZ throughout sleep‐wake cycles by recording GRAB ATP1.0 and GRAB Ado1.0 signals. H) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, EMG traces, representative GRAB ATP1.0 (ATP) and GRAB Ado1.0 (Ado) fluorescence traces (z‐score) during sleep‐wake cycles; color code indicates NREM sleep, REM sleep, and wakefulness. I) Mean (±s.e.m.) ΔF/F (z‐score) of GRAB ATP1.0 (ATP) signals during each state. n = 3 mice. A one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0080 (Wake vs REM), * p = 0.0390 (NREM vs REM). J) Correlation analysis of GRAB ATP1.0 and GRAB Ado1.0 signals (left) and the shuffled control (right). n = 31 trials, recorded from 3 individuals. Pearson correlation analysis, * p = 0.0152, r = 0.4323, red line shows linear regression line (Y = 0.2824*X+1.788), dot line indicates 95% regression range. K) Setup for optogenetic activation of astrocytes in the PZ following the administration of ARL67156 (CD73 inhibitor), NBTI (ENT inhibitor), or vehicle. L) Representative image of the expression of AAV‐GfaABC1D‐ChrimsonR‐mCherry in the PZ and the location of bilateral canula. Scale bar, 100 µm; red, ChrimsonR; blue, DAPI. M‐N) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of ARL67156 ( n = 5 mice), NBTI ( n = 5 mice), or vehicle ( n = 5 mice). Two‐way ANOVA test, Tukey's multiple comparisons test. From NREM to Wake: ** p = 0.0029 (Vehicle+OPTO vs ARL67156+OPTO), ** p = 0.0044 (ARL67156+OPTO vs NBTI+OPTO); From NREM to NREM, ** p = 0.0029 (Vehicle + OPTO vs ARL67156+OPTO), ** p = 0.0026 (ARL67156+OPTO vs NBTI+OPTO). **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Generated, Binding Assay, Expressing, Immunohistochemistry, Staining, Activation Assay, Fluorescence, Control