Regulation of CTCF expression, its effect on islet function, and CUT&Tag analysis of CTCF genome-wide binding profiles. (a) Immunoblot of total islet protein lysates from mice fed with HFD for 6 mo and their corresponding controls (n = 3 biological triplicates). (b) Immunoblot of total protein lysates from Min6 cells transduced with spCas9 and sgRNA expressing lentivirus. Cells transduced with lentivirus expressing only spCas9 were used as the control group (CTR). (c) Immunoblot of total islet protein lysates from mice fed with HFD for 2 mo and their corresponding controls (n = 3 biological triplicates). (d) Dynamic glucose-stimulated insulin secretion (left panel) and biphasic glucose-stimulated insulin release levels (right panel) of mouse islets treated with TNFα (50 ng/ml) and PA (0.5 mM) for 24 h. Each islet sample was pooled from at least three animals. Data represent mean ± SD; n = 3 technical replicates; ***, P < 0.001; two-tailed unpaired Student’s t test. (e) ATP/ADP ratio of CTCF or GFP expressing mouse islets under low-glucose (2.8 mM) or high-glucose (16.8 mM) conditions. Islets were treated with TNFα (50 ng/ml) and PA (0.5 mM) for 24 h prior to glucose stimulation. Each sample was pooled from at least three animals. Data represent mean ± SEM (n = 4 replicates of islet samples, each sample represents 20 IEQ islets); ***, P < 0.001; two-tailed unpaired Student’s t test. (f) Metagene heatmap of the genome-wide CTCF CUT&Tag peaks’ occupation in the −5 to +5 kb regions flanking the TSS in islets from indicated groups. In each treatment, CUT&Tag libraries were prepared using islets pooled from three mice, with 2–3 technical replicates. The scale bar indicates peak density. (g) Known motif analysis within reversible CTCF binding peaks. Consensus non-redundant motifs (Motif), transcriptional factor names (Name), and P values are shown. Data in a–e are representative of two independent experiments. Source data are available for this figure: SourceData FS5.