Fig 2: EPB41L4A-AS1 negatively regulates GLUT4 transcription by increasing H3K27 crotonylation and PGC1-ß lysine acetylation. As shown, EAS1 represents EPB41L4A-AS1; (A-B) Pearson's correlation analysis of TP53 or EAS1 and GLUT4 (SLC2A4) expression in human muscle and adipose tissues. Data were obtained from the GTEx RNA-seq database, and images were obtained from the GEPIA website. (C) GLUT4 mRNA levels measured using qPCR after the over-expression of EAS1/EAS1-ATG-mut for 48 h or after the stable knockdown of EAS1 (n = 3), Student's t-test. (D) Primer locations in the GLUT4 promoter, as designed for chromatin immunoprecipitation-qPCR. (E) H3K27ac enrichment in the GLUT4 promoter in A673 cells with EAS1/EAS1-ATG-mut over-expression or EAS1 knockdown (n = 3). Data shown as log2(fold change); EAS1 and EAS1-ATG-mut expression was normalized to that of the vector (control, value = 0), whereas shEAS1 expression was normalized to that of shNC (control, value = 0). (F) H3K14ac enrichment in the GLUT4 promoter in A673 cells after the over-expression of EAS1 or EAS1-ATG-mut plasmids for 48 h and in A673 cells with stable EAS1 knockdown (n = 3). (G) Total H3K27cr levels were measured by immunoblotting using stable EAS1 knock-down A673 cells or A673 cells with EAS1 or EAS1-ATG-mut over-expression (n = 3). (H) Interaction between EPB41L4A-AS1 and H3K27cr (n = 3). (I) H3K27cr enrichment in the GLUT4 promoter was measured with the ChIP assay (n = 3). (J-K) A673 cells were incubated with 10 mM crotonate or at high glucose concentrations or both for 24 h, and qRT-PCR was conducted to measure the EPB41L4A-AS1 (J) and GLUT4 levels (K) (n = 3). (L) shNC and shEAS1 A673 cells were incubated with or without glucose at high concentrations and 10 mM crotonate for 24 h, and ChIP-qPCR was conducted to detect the occupation of H3K27cr in the GLUT4 promoter region (n = 3). (M-N) Immunoprecipitation for measuring the level of lysine acetylation in PGC1-ß in A673 cells (n = 3). (O-Q) GCN5 and PGC1-ß interactions were analyzed by the immunoprecipitation assay in stable EAS1 knock-down A673 cells (n = 3) and EAS1-ATG-mut-over-expressing A673 cells (n = 3)

Fig 3: EPB41L4A-AS1 regulates glucose uptake through GLUT2 or GLUT4. As shown, EAS1 represents EPB41L4A-AS1; (A-D) GLUT2 protein level assayed by western blotting of proteins from HepG2 (n = 3) or L02 (n = 3) cells with transient EPB41L4A-AS1/EPB41L4A-AS1-ATG-mut over-expression or stable EPB41L4A-AS1 knockdown, Student's t-test. (E-F) Cell membrane surface GLUT2 protein levels in HepG2 (n = 3) or L02 (n = 3) cells stimulated with 100 nM insulin for 30 min after the transient over-expression of EPB41L4A-AS1/EPB41L4A-AS1-ATG-mut for 48 h or after the stable knockdown of EPB41L4A-AS1, as measured by using western blotting. (G-H) Total GLUT4 protein level (n = 3) and cell membrane surface GLUT4 protein level (n = 3) in A673 cells stimulated with 100 nM insulin for 30 min after the transient over-expression of EPB41L4A-AS1/EPB41L4A-AS1-ATG-mut for 48 h or after the stable knockdown of EPB41L4A-AS1, as measured using western blotting

Fig 4: EPB41L4A-AS1 regulates TXNIP transcription via enhancement of H3K14 and H3K27 acetylation. As shown, EAS1 represents EPB41L4A-AS1; (A) TXNIP and EPB41L4A-AS1 expression correlation in muscle and liver tissues; data obtained from GTEx database and the GEPIA website are plotted in the diagram. (B-D) The mRNA expression of TXNIP (n = 3) measured using qRT-PCR. (E-F) TXNIP protein levels in HepG2 (n = 3), A673 (n = 3),and L02 (n = 3) cells after the stable knockdown of EPB41L4A-AS1 or the transient over-expression of EPB41L4A-AS1 or EPB41L4A-AS1-ATG-mut, as measured using western blotting. (G) Schematic representation of the primer location in the TXNIP promoter region. (H) H3K27cr enrichment in the TXNIP promoter measured by chromatin immunoprecipitation (ChIP)-qPCR in HepG2 cells over-expressing EPB41L4A-AS1 or EPB41L4A-AS1-ATG-mut or with EPB41L4A-AS1 knockdown (n = 3), Student's t-test. (I-J) H3K27ac and H3K14ac enrichment in the TXNIP promoter of A673 cells after the over-expression of EPB41L4A-AS1 or EPB41L4A-AS1-ATG-mut or the stable knockdown of EPB41L4A-AS1 (n = 3), as evaluated using ChIP-qPCR analysis, Student's t-test. (K-L) GCN5 was transiently knocked down in A673 cells for 48 h using GCN5-specific siRNAs, and the GCN5 and TXNIP protein and mRNA levels were measured by immunoblotting (n = 3) and qPCR (n = 3), respectively. (M) GCN5 occupation in the TXNIP promoter in A673 cells (n = 3) was assayed by ChIP-qPCR. (N) The interaction between MLXIP and GCN5 in A673 cells. (O) RNA immunoprecipitation-qPCR analysis of the interaction between EPB41L4A-AS1 and MLXIP in A673 cells (n = 3). (P) MLXIP and TXNIP RNA expression correlation determined by Pearson's correlation analysis in muscle and liver tissues. Data were obtained from the GTEx database, and the diagram was obtained from the GEPIA website. (Q) MLXIP enrichment at the TXNIP promoter in A673 cells after the stable knockdown of EPB41L4A-AS1 or over-expression of EPB41L4A-AS1 or EPB41L4A-AS1-ATG-mut (n = 3), as detected using ChIP-qPCR

Fig 5: EPB41L4A-AS1 is up-regulated in patients with type 2 diabetes mellitus (T2DM) and T2DM cell models. (A-B) The expression of EPB41L4A-AS1 in the liver (n = 10) and muscle (n = 16) tissues of patients with T2DM or insulin resistance compared with that in normal controls. Data are from the GEO databases GSE23343 and GSE6798 datasets, Mann–Whitney U test. (C) Glucose uptake in cells before and after treatment at high glucose concentrations (n = 3). (D) Glucose consumption in cells treated at normal glucose concentrations for 6 h after pre-treatment with high- or normal-glucose DMEM (n = 3). (E-H) EPB41L4A-AS1 was measured in cells treated at high glucose concentrations for 0, 0.25, 0.5, 2, 12 and 24 h (n = 3). One-way analysis of variance, followed by Tukey's post-hoc test. (I-J) The expression of EPB41L4A-AS1 in cells incubated with 5 mM glucosamine for 24 h (n = 3)