Fig. 6: HuR targets and stabilizes Insig1.
From: The RNA-binding protein HuR is a negative regulator in adipogenesis
a Western blot to confirm the presence of HuR protein in the IP sample by anti-HuR. b Circle plot depicting the top 10 most significant biological processes generated using the top 200 most enriched HuR-binding genes in Ctrl. The inner circle is colored by the significance of the GO process. The outercircle represents the scatterplot of the enrichment (log2(Ctrl/HuR-FKO)) for all the genes found within each GO term. c The accumulative fraction curves were plotted for the fold changes of targets and other genes between the FKO and control samples. Kolmogorov–Smirnov test. d Real-time PCR to confirm the expression of Insig1 in BAT, iWAT, and eWAT (Ctrl: n = 6; HuR-FKO: n = 8). e RIP assay with anti-HuR in epididymal white adipose tissue lysate from the HuR-FKO and control littermates to examine the amount of Insig1 mRNA in the IP samples. Fabp4 was used as a control. 5% tissue lysate in the IP reaction was used as input (Ctrl, n = 3; HuR-FKO, n = 6). f RNA pull-down assay for the HuR-recognizing RNA segments in Insig1 3′UTR (detailed in Methods). g The diagram of a psiCHECK2 reporter with a full length Insig1 3′UTR (upper) or a mutated 3′UTR without the two HuR-binding sites (lower). h, i The plasmid diagrammed in (g) was co-transfected with HuR overexpression plasmid or control (XZ201) into 293 cells. Actonmycin D was added to stop the transcription, followed by real-time PCR for h hRluc, i hLuc, and j 18 s during a time course. The remaining fractions of hRluc mRNA at each point were fit into a first-phase decay curve to derive the RNA half-life. n = 6 per group. k Primary white preadipocytes were isolated from HuR-KFO and control animals for culture and then induced to differentiate for 5 days. Actinomycin D was added to track the decay rate. l 18S mRNA was used a control. n = 5 per group. Error bars are mean ± SEM. Statistical significance was determined by Student’s t-test; *p < 0.05.
