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. 2023 Jun 15;26(7):107128. doi: 10.1016/j.isci.2023.107128

Figure 4.

Figure 4

Effect of liver metabolic state on CTCF differential expression

(A and B) Protein expression of CTCF in ad-lib fed (8 a.m.) and 24 h fasted (8 a.m. onwards) mice liver (A) Representative immunoblots and (B) quantification of CTCF band intensities. CTCF band intensities were quantified with tubulin as a loading control and presented as fold change in comparison to ad-lib fed (N = 5, n = 3–4).

(C) mRNA expression of Ctcf in ad-lib fed (8 a.m.) and 24 h starved liver (8 a.m. onwards), normalized to H2a transcript levels and plotted as fold change with respect to ad-lib fed (N = 3, n = 3).

(D and E) Immunofluorescence (D, Representative images) and (E) quantifications of CTCF in ad-lib fed and 24 h starved liver sections, captured at 10× magnification and optically zoomed (3×) (Top, Right) (scale bar depicts 100μm) (For E, n = 6).

(F) Protein expression of CTCF in primary hepatocytes treated with high glucose (HG, 25mM glucose) and glucose-free (NG) media for 16 hrs.

(G and H) Immunofluorescence (G, Representative images) and (H) quantifications of CTCF in HG and NG treated primary hepatocytes, captured at 60× magnification (scale bar depicts 5μm) (For H, N = 2, n = 60 nuclei).

(I) Genome Browser (IGV) snapshots of H3K27ac ChIP-seq in ad-lib fed and 24 h starved liver at Ctcf locus.

(J) Genome Browser snapshot of CREB and PPARα ChIP-seq peaks from starved mice liver. Blue boxes are depicting promoter-specific histone marks at Ctcf locus (H3K9ac and H3K4me3 ChIP-seq (Encode Database) in liver).

(K and L) Relative Ctcf mRNA expression in primary hepatocytes treated with (K) HG, low glucose (LG, 5mM glucose) and 10μM forskolin (Fsk) supplemented in LG media for 12 h, normalized with H2a transcript levels and plotted as fold change compared to LG treatment (N = 2, n = 3–6) and (L) HG and 50 μM WY14643 supplemented in HG media for 10 h, normalized with actin (Actb) and represented as fold change compared to HG treatment (N = 2, n = 3). Data are represented as mean ± SEM. For B, C, E, H, K & L statistical significance was calculated by using a Student’s t test (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001).