Fig 2: Possible involvement of adiponectin in the myocardium after 14 days of cocaine administration. Protein expressions of adiponectin (Adipo), AMP-activated protein kinase (AMPK), and phosphorylated AMPK (p-AMPK) in left ventricles from the control and cocaine groups. Levels of actin served as the internal control. Individual values are presented in black bullets. Each bar represents mean and S.D. *, p < 0.05.

Fig 3: Treatments with Alchemilla monticola Opiz. (ALM) extract, astragalin (AST) and quercitrin (QUE) suppressed adipogenic gene expression in human SGBS adipocytes. Relative mRNA expression normalized to vehicle control group (??Cq) for acetyl-coA-carboxylase ( ACC; A), adiponectin ( ADIPOQ; B), CCAAT/enhancer-binding protein alpha ( CEBPA; C), fatty acid synthase ( FASN; D), peroxisome proliferator-activated receptor gamma ( PPARG; E) and sterol regulatory element-binding protein ( SREBP; F) from the RT-qPCR analysis. Three replicates were performed from three independent biological repeats. Data are presented as mean ± SEM. *p < 0.05 versus the vehicle control group.

Fig 4: Generation of conditional Ube2i gene deletion mice. (A)Ube2ifl/fl mice were generated using CRISPR/Cas9 gene editing. Exons 3 and 4 of the Ube2i (Ubc9) gene were targeted by sgRNAs designed complementary to intronic sequences flanking the exons, then loxP sequences were introduced by DNA donor oligonucleotides. LoxP sites were inserted before exon 3 and after exon 4 (black arrows). Primers detect the 5’ (P1, P2) and 3’ (P3, P4) loxP sequences. (B) PCR analyses of floxed alleles at the targeted loci in genomic DNA extracted from ear clips of wild-type (+/+), fl/+, and fl/fl mice. PCR products were run on agarose gels with expected band sizes for P1–P2: wild-type (+) 319 bp and loxP allele (fl) 353 bp and P3–P4: wild-type (+) 427 bp and loxP allele (fl) 461 bp. The image shows a wild-type control #1107 +/+, founder heterozygous mouse #974 fl/+, and homozygous F2 offspring #1091 fl/fl. (C) Western blot analysis of UBE2I expression in inguinal WAT (iWAT) derived fibroblasts from Ube2ifl/fl mice after transduction with adenoviral GFP or Cre. To determine cell autonomous effects of Ube2i deletion in adipocytes, tamoxifen inducible Cre-expressing mice (CAG-Cre) were crossed with Ube2ifl/fl mice. iWAT SVF cells were isolated from CAG-Cre;Ube2ifl/fl and Ube2ifl/fl control mice. All cells were treated with tamoxifen to induce Cre recombination followed by adipocyte differentiation (diff) for eight days. (D) Western blot analysis and (E) relative gene expression by qPCR were performed to assess Ube2i depletion, adipocyte maturation, and beige fat cell markers. Gray = Ube2ifl/fl, yellow = CAG-Cre;Ube2ifl/fl. Data are presented as mean +/- SEM, *p < 0.05. (F) Brightfield and fluorescent images of differentiated Ube2ifl/fl and CAG-Cre;Ube2ifl/fl cells stained for lipids (green, LipidTOX), perilipin (red), and nuclei (blue, DAPI). Scale bars 50 µm. (G) Whole cell lysates from differentiated cells were subjected to Western blot analysis of cleaved (p41/43, p18) and uncleaved Caspase-8. (H) Strategy for generating adipocyte-specific Ube2i gene deletion. Ube2ifl/fl mice were bred with Adipoq-Cre mice to generate adipocyte-specific Ube2i knockout (Ube2ia-KO) and Ube2ifl/fl (control) mice. (I) To validate gene deletion of Ube2i, genomic DNA was extracted from Ube2ia-KO and Ube2ifl/fl gonadal WAT (gWAT) samples and PCR products were run on an agarose gel to detect the 5’ (P1, P2 primers) and 3’ (P3, P4 primers) loxP sequences, as well as a product that spans exons 3–4 (P1+P2+P4; 1597 bp, green arrow) or the deletion product (509 bp, red arrow). (J) Western blots of whole tissue lysates from iWAT and BAT of seven-day-old mice were probed for UBE2I and adiponectin (ADIPOQ), and HSP90 served as the invariant loading control.

Fig 5: CRISPR/Cas9-mediated NPR3 ablation increases insulin-stimulated glucose uptake and de novo lipogenesis. a Representative Western blot for NPR3 (60 kD) and adiponectin (AdipoQ) in SGBS pre-adipocytes (d0) and adipocytes (d14) in control cells (EV) and NPR3 KO cells with densitometric analysis displayed as mean and SEM of 4 independent experiments in relation to tubulin. b Differentiation rates of adipocyte cultures (adipocytes/total cells). c Insulin-dependent glucose uptake was measured by scintillation counting and normalized to the basal rate of control cells (0 nM insulin). d Insulin-dependent de novo lipogenesis was measured by scintillation counting and normalized to the basal rate of control cells (0 nM insulin). e Insulin-dependent glucose uptake in control (EV) and NPR3 KO cells transfected with non-targeting (NT) or miR-146a mimic was measured by scintillation counting and normalized to the basal rate of EV cells (0 nM insulin). Data are displayed as mean and SEM of 5 (c) and 4 (d) independent experiments. Statistics: (a) two-way ANOVA with Bonferroni correction (c and d) two-way ANOVA with Bonferroni correction and non-linear fit with three parameters. *p < 0.05, **p < 0.01, ****p < 0.0001