Fig. 2

In vitro Lep UE hypomethylation and effect on adipocyte differentiation. A Principal scheme of targeted in vitro hypomethylation using a dCas9-Suntag-TET1 system showing in the top the ‘all in one’ vector which contains dCas9 peptide array (linker length: 22 amino acids), antibody-sfGFP-TET1CD, and gRNA expression system. Scheme was generated using biorender.com. B Workflow of the experiment, which is described in detail in the methods section. For the hypomethylation, epididymal preadipocytes were transfected by electroporation with all-in-one vectors including three different gRNAs targeting the Lep UE (= Lep UE gRNA, red). For control, we used untreated cells (= UT, dark grey) and cells transfected with the all-in-one vector without gRNA (= plasmid control, light grey). Workflow was generated using biorender.com. C Boxplot shows DNA methylation level [%] of Lep UE across all three analysed CpG sites by bisulphite pyrosequencing. Significance of differences between treatments was calculated using Kruskal–Wallis test, followed by pairwise comparisons by Wilcoxon rank-sum test corrected for multiple testing by FDR (***FDR < 0.001, ns FDR > 0.05). Dots represent mean methylation levels of days 0, 2, 4, 6, and 8 of differentiation for each experiment (n = 3 experiments × 5 time points). D Barplot shows the mean of log transformed Lep mRNA levels normalised to Rplp0 mRNA levels for days 4, 6, and 8 of adipocyte differentiation from n = 3 experiments. Results of mixed two-way ANOVA used to assess the effect of treatment and time on Lep expression are shown on top of the graph. Pairwise comparisons of treatment effects using paired Student's t-test (paired by experiment number) are depicted in the graph. No significant differences were found after correction for multiple testing by FDR. Shown significance level are uncorrected for multiple testing: #P < 0.05. E Barplot shows the lipid amounts in cells during differentiation on days 0, 4, and 8 between treatments. Shown is the average ratio of Adipored and Hoechst fluorescence intensity from n = 12 wells in n = 3 experiments. Results of mixed two-way ANOVA used to assess the effect of treatment and time on lipid accumulation are shown on top of the graph. Significance levels from pairwise comparison of time points using paired Student's t-test (paired by experiment number) and corrected for multiple testing by FDR are depicted in the graph (***FDR < 0.001, **FDR < 0.01, and *FDR < 0.05). No significant differences are found when comparing treatments