Fig 1: Fbxl19 is a conserved miR-26 target that promotes adipogenesis. (A) Experimental design to identify candidate miR-26 targets that regulate adipogenesis. RNA-seq was used to identify (1) genes up-regulated in miR-26-TKO vs. wild-type SVF cultures and (2) genes down-regulated in dox-treated versus untreated SVF cultures from miR-26a transgenic mice (M2rtTA; miR-26a). Genes were further filtered for Targetscan7.2-predicted miR-26 targets (http://www.targetscan.org). (B,C) qRT-PCR analysis of Fbxl19 expression in wild-type and miR-26-TKO SVFs (B) and miR-26a transgenic SVFs with or without dox treatment (C). n = 3 biological replicates. (D) Western blot of FBXL19 protein levels in wild-type and miR-26-TKO SVFs. Data are representative of three independent experiments. α-tubulin (TUB) represents a loading control. (E) Representative images of Oil Red O-stained in vitro differentiated wild-type or miR-26-TKO SVF cultures following siRNA knockdown of Fbxl19. siNT, nontargeting control siRNA. (F) qRT-PCR analysis of adipogenic gene expression in SVF cultures in E. (G) Western blot of Flag-tagged mouse FBXL19 or PPARγ2 in transfected wild-type SVF cultures. (H) Representative images of Oil Red O-stained SVF cultures after overexpression of the indicated proteins. (I) qRT-PCR analysis of adipogenic gene expression in SVF cultures with GFP or FBXL19 overexpression. (J) Schematic representation of conserved miR-26 binding sites in the 3′ UTR of Fbxl19. Mutations introduced to disrupt miR-26-binding are shown below the alignment and highlighted in red. (K) Relative firefly luciferase activity of reporter constructs containing wild-type or mutant miR-26-binding sites from the Fbxl19 3′ UTR following cotransfection with control or miR-26a mimics. Data represent the average of three independent experiments, each performed with technical triplicates. Data represented as mean ± SD (*) P < 0.05; (**) P < 0.01, calculated using two-tailed t-test.
Fig 2: miR-26 suppresses adipocyte progenitor cell differentiation. miR-26 functions within the APC lineage to inhibit mobilization and subsequent adipocyte production by down-regulating FBXL19, a novel driver of adipogenesis.
Fig 3: FBXL19 binds to FOXM1 and inhibits FOXM1 protein level. A The binding of FBXL19 to FOXM1 in SPN mouse lung tissue was tested by the Co-IP assay; B Protein levels of FOXM1 in lung tissues were measured by Western blot assay, N = 6. Experiments were repeated 3 times. Data in panel B were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test
Fig 4: FBXL19 overexpression attenuates Spn-induced lung injury in pneumonia immature mice. Immature mice were intravenously injected with FBXL19 lentivirus overexpression vectors (FBXL19), with lentivirus empty vector (NC) as the negative control. Then, a pneumonia immature model (SPN) was established by Spn induction. A–B: FBXL19 expression levels in lung tissues were determined by RT-qPCR (A) and Western blot assay (B); C The survival rates of immature mice within 14 days were analyzed by Kaplan–Meier plots; D Bacterial loads in BALF in each group; E Lung injury scoring and representative images of H&E staining; F The lung wet/dry weight ratios; G MPO activity in lung tissues; H Contents of IL-1β, IL-6, and IL-10 in lung tissues examined by ELISA. C N = 18, A, B and D–H N = 6. Experiments were repeated 3 times. Data in panel C were analyzed using the log-rank test, data in panel D were analyzed by the t test, and data in panels A, B, E, F, G, and H were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test. BALF broncho-alveolar lavage fluid, MPO myeloperoxidase
Fig 5: FBXL19 ubiquitinates and degrades FOXM1. A Ubiquitination levels of FOXM1 in lung tissues; B: The binding of FBXL19 to FOXM1 was tested by the Co-IP assay; MLE-12 cells were infected with FBXL19 lentivirus overexpression vectors (FBXL19), with lentivirus empty vector (NC) as the negative control; C, D FBXL19 expression levels in cells were determined by RT-qPCR (C) and Western blot assay (D); MLE-12 cells were treated with MG132, with dimethylsulfoxide (DMSO) as the negative control; E Ubiquitination levels of FOXM1 in cells; F–G FOXM1 expression levels in cells were determined by Western blot assay (F) and RT-qPCR (G). Experiments were performed in triplicate. Data in panel C were analyzed using the t test, and data in panels D, F, and G were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test
Supplier Page from Abcam for Anti-FBXL19 antibody [EPR11957]