Journal: Human Genetics and Genomics Advances

Article Title: Using a modular massively parallel reporter assay to discover context-dependent regulatory activity in type 2 diabetes-linked noncoding regions

doi: 10.1016/j.xhgg.2026.100606

Figure Lengend Snippet: HNF1 transcription factor motifs contribute to enhancer activity near selected T2D-associated variants (A) For 47 HNF1-motif-overlapping fragments with significant INS promoter-bias effects on activity, we designed three versions: original (motif intact), deleted (motif removed and sequence adjusted), and shuffled (dinucleotide-shuffled motif). When the tested variant was adjacent to the motif, we synthesized both reference and alternative alleles for each version. For variants directly overlapping the motif, we generated only one deletion and one shuffled fragment. (B) We synthesized four fragments corresponding to the variant rs1635852, which overlaps an HNF1 motif at a high-information-content position. The T2D risk allele (T) disrupts this motif, while the non-risk allele (C) matches the consensus. (C) Shuffling the motif significantly decreased enhancer activity compared to intact fragments with either the risk T (Wilcoxon rank-sum test p = 0.016) or non-risk C ( p = 0.008) allele. Motif deletion also significantly decreased enhancer activity compared to the non-risk C allele ( p = 0.016). (D) For the variant rs11819995, located 11 bp upstream of an HNF1 motif, we synthesized six fragments. (E) Deletion of the motif significantly decreased enhancer activity for both the reference (C, non-risk) and alternative (T, risk) alleles ( p = 0.008 for both alleles). Shuffling the motif likewise reduced activity for both alleles ( p = 0.008 for the reference allele and p = 0.056 for the alternative allele). (F) To assess context-specific effects, we cloned these fragments into MPRA vectors with the SCP1 or skeletal-muscle-specific MYBPC2 promoter and delivered all three to LHCN-M2 human skeletal muscle myotubes ( n = 6). (G) When paired with the INS promoter, the shuffled rs11819995-containing fragment showed increased activity relative to the original fragment ( p = 0.015); however, none of the fragments containing rs11819995 functioned as enhancers in LHCN-M2 myotubes, regardless of promoter context. Overall, their activity is highest when paired with the skeletal-muscle-specific promoter.

Article Snippet: We obtained INS-1 832/13 rat insulinoma cells from Dr. Christopher Newgard (Sarah W. Stedman Nutrition and Metabolism Center, Duke University, Durham, NC) and LHCN-M2 human skeletal muscle myoblasts from Evercyte.

Techniques: Activity Assay, Sequencing, Variant Assay, Synthesized, Generated, Clone Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Flavonoids from Elsholtzia ciliata restore redox electron flow and metabolic signaling via PTP1B inhibition in muscle and liver cells

doi: 10.1080/14756366.2026.2666369

Figure Lengend Snippet: Principal Component Analysis (PCA) biplots visualise the distinct mechanistic clustering of E. ciliata -derived compounds and reference drugs across all in vitro models. PCA biplots were generated from the combined functional (glucose uptake) and gene expression (qRT-PCR) data for each of the four models: (A) C2C12-Insulin, (B) C2C12-Leptin, (C) C2C12-Adiponectin, and (D) Hepa1c1c7-PA con. Data points (x) represent individual biological replicates, and the ellipses represent the 95% confidence intervals for each treatment group. Loading vectors (black arrows) indicate the direction and magnitude of influence for each variable (e.g. Ptpn1 , Ampka1 , Glucose uptake) on the principal components (PC1 and PC2). The percentage of variance explained by each principal component is shown on the axes. The plots visually confirm the clear separation of the disease control groups (PA-treated) and the distinct mechanistic clusters formed by EC2, EC5, Met, and NAC.

Article Snippet: Murine C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) .

Techniques: Derivative Assay, In Vitro, Generated, Functional Assay, Gene Expression, Quantitative RT-PCR, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Flavonoids from Elsholtzia ciliata restore redox electron flow and metabolic signaling via PTP1B inhibition in muscle and liver cells

doi: 10.1080/14756366.2026.2666369

Figure Lengend Snippet: Baseline glucose uptake and metabolic gene expression in differentiated C2C12 myotubes under non-PA conditions. Differentiated C2C12 myotubes were treated for 24 h with each of metabolic hormones (insulin, leptin, or adiponectin), reference antidiabetic agents (Met, Duo, NAC), or E. ciliata -derived compounds (EC2–EC5). (A) Glucose uptake (%) was measured in hormone/antidiabetic agent-treated cells (top) and EC-treated cells (bottom). (B) Relative levels of gene expression of Ptpn1 , Pparg , Ampka1 , Ampka2 , and Txnip was assessed via qRT-PCR. ECs were treated at the higher dose (20 μM). The concentration of ECs used in this experiment was 10 μM (L) or 20 μM (H). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. non-treated (vehicle)control. PA , palmitate; Ins-C , insulin- control; Lep-C, leptin-control; Adi-C, Adiponectin-control ; ScI, sc-222227; UA, ursolic acid; Met, metformin; Duo, Duoglow (pioglitazone + dapagliflozin); NAC, N-acetylcysteine; EC2–EC5, E. ciliata -derived compounds; Ptpn1 , protein tyrosine phosphatase 1B; Pparg , peroxisome proliferator-activated receptor gamma; Ampka1/2 , AMP-activated protein kinase alpha 1/2; Glut4 , glucose transporter 4.

Article Snippet: Murine C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) .

Techniques: Gene Expression, Derivative Assay, Quantitative RT-PCR, Concentration Assay, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Flavonoids from Elsholtzia ciliata restore redox electron flow and metabolic signaling via PTP1B inhibition in muscle and liver cells

doi: 10.1080/14756366.2026.2666369

Figure Lengend Snippet: Restoration of the impairment of glucose uptake by E. ciliata -derived compounds in PA-induced insulin-resistant C2C12 myotubes. (A) Glucose uptake levels (%) were quantified. (B) Relative levels of mRNA expression of Ptpn1 , Glut4 , Pi3k , and Akt were analysed via qRT-PCR. EC2-H and EC5-H downregulated Ptpn1 and restored Glut4 levels. ECs were treated at the dose of 20 μM. The concentration of ECs used in this experiment was 10 μM (L) or 20 μM (H). Data are presented as mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01 vs. Insulin + PA control group. † p < 0.05, †† p < 0.01 vs. insulin control group. PA, palmitate; Ins-C, insulin-only control; Ins-PA, insulin + PA; ScI, sc-222227; UA, ursolic acid; Met, metformin; Duo, Duoglow (pioglitazone + dapagliflozin); NAC, N-acetylcysteine; EC2–EC5, E. ciliata -derived compounds; Ptpn1 , protein tyrosine phosphatase 1B; Glut4 , glucose transporter 4; Pi3k , phosphoinositide 3-kinase; Akt , protein kinase B.

Article Snippet: Murine C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) .

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Concentration Assay, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Flavonoids from Elsholtzia ciliata restore redox electron flow and metabolic signaling via PTP1B inhibition in muscle and liver cells

doi: 10.1080/14756366.2026.2666369

Figure Lengend Snippet: Reversal of leptin resistance by E. ciliata -derived compounds in PA-treated C2C12 myotubes. Glucose uptake in response to leptin stimulation. Glucose uptake was expressed as a percentage of normal cells. (B) Gene expression analysis of Ptpn1 , Jak2 , Stat3 , and Glut4 was performed by qRT-PCR. ECs were treated at the dose of 20 μM. Data are presented as mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01 vs. Lep + PA control group. † p < 0.05, †† p < 0.01 vs. Leptin control group. PA, palmitate; Lep-C, leptin-only control; Lep-PA, leptin + PA; ScI, sc-222227; UA, ursolic acid; Met, metformin; Duo, Duoglow (pioglitazone + dapagliflozin); NAC, N-acetylcysteine; EC2–EC5, E. ciliata -derived compounds; Ptpn1 , protein tyrosine phosphatase 1B; Jak2 , Janus kinase 2; Stat3 , signal transducer and activator of transcription 3; Glut4 , glucose transporter 4.

Article Snippet: Murine C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) .

Techniques: Derivative Assay, Gene Expression, Quantitative RT-PCR, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Flavonoids from Elsholtzia ciliata restore redox electron flow and metabolic signaling via PTP1B inhibition in muscle and liver cells

doi: 10.1080/14756366.2026.2666369

Figure Lengend Snippet: Effects of E. ciliata -derived compounds on redox homeostasis: NAD⁺/NADH, NADP⁺/NADPH ratios, and FAD levels. C2C12 myotubes were co-treated with palmitate (PA, 300 μM for 48 h) and each test compound or hormone for the final 24 h. The NAD⁺/NADH ratio (A), NADP⁺/NADPH ratio (B), and FAD (C) levels were measured to assess redox homeostasis. Data are presented as mean ± SEM ( n = 5). * p < 0.05, ** p < 0.01 vs. corresponding –PA control group (i.e. basal); † p < 0.05, †† p < 0.01 vs. PA-treated (vehicle) control group. PA, palmitate; ScI, sc-222227; UA, ursolic acid; Met, metformin; Duo, Duoglow (pioglitazone + dapagliflozin); NAC, N-acetylcysteine; EC2–EC5, E. ciliata -derived compounds; NAD⁺, nicotinamide adenine dinucleotide (oxidised form); NADH, nicotinamide adenine dinucleotide (reduced form); NADP⁺, nicotinamide adenine dinucleotide phosphate (oxidised form); NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); FAD, flavin adenine dinucleotide.

Article Snippet: Murine C2C12 myoblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) .

Techniques: Derivative Assay, Control