Fig 2: Effects of TL1A on adipogenic differentiation of MEFs and 3T3-L1 cells.Two days post-confluent MEFs or 3T3-L1 cells were cultured in adipogenic induction medium supplemented with TL1A recombinant protein at the specified concentrations (0, 10, 20, 50, 100, 200 ng/mL). (A) Oil Red O staining was conducted after adipogenic induction for 7 days. (B) The average optical density (OD) was measured using a Universal Microplate Spectrophotometer at 490 nm. (C) Measurement of triglyceride (TG) content in MEFs 7 days after adipogenic differentiation. (D-E) Both Oil Red O staining and triglyceride assays in 3T3-L1 cells were performed on day 7 of differentiation. (F-G) qRT-PCR analysis was conducted to evaluate the expression of adipogenic markers aP2, Plin1, Plin2 and PPARγ in 3T3-L1 cells 7 days after adipogenic differentiation. (H) qRT-PCR analysis was conducted to evaluate the expression of VEGFA and several adipogenic markers C/EBPα/β/δ, PPARγ2, aP2, CD36, Plin1, Plin2, adispin, Glut4 and LPL in MEFs 7 days after adipogenic differentiation. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the group of adipocytes without TL1A treatment (n = 3).

Fig 3: qRT-PCR analysis of adipogenesis regulators during differentiation induction.After two days post-confluence, MEFs were treated with standard differentiation medium and TL1A recombinant protein (0, 10, 20, 50, 100, 200 ng/mL). At specified time points, cells were harvested for RNA isolation and subsequent qRT-PCR analysis. *P < 0.05 vs. the group of adipocytes without TL1A treatment.

Fig 4: TL1A induces adipogenic markers expression during adipocyte differentiation.(A-D) MEFs or 3T3-L1 cells were induced to undergo adipocyte differentiation for 3 days in the presence of the specified concentrations of TL1A. Total protein was extracted to determine C/EBPα, C/EBPβ, PPARγ2, PPARγ1 and aP2 protein expression by Western blotting with quantitation of band density. (E-H) MEFs or 3T3-L1 cells were induced to undergo adipocyte differentiation for 7 days with the indicated concentrations of TL1A. Total protein was extracted to determine PPARγ2, PPARγ1, CD36 and aP2 protein expression by Western blottingwith quantitation of band density. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the group of adipocytes without TL1A treatment (n = 3).

Fig 5: TL1A inhibits β-catenin expression by promoting the phosphorylation of YAP1.MEFs were treated with standard differentiation medium and TL1A at the specified concentrations (0, 10, 20, 50, 100, 200 ng/mL) for 7 days. (A) Total cellular proteins were extracted, and the expression levels of β-catenin, phosphorylated YAP1S127 (p-YAP1S127) and total YAP1 proteins were determined by Western blotting. (B) The relative amounts of β-catenin protein expression were calculated according to the grayscale values and were showed in the histogram. (C) The quantitation of ratio (p-YAP1S127/total YAP1) to reflect the inactivation of YAP1 signaling pathway. (D-E) At the end of adipogenesis induction, protein expression of phosphorylated β-catenin (p- β-catenin) and YAP1 in the cytoplasm extract from MEFs was determined by Western blotting. (F-G) Nuclear extracts were isolated from the MEFs and analyzed for protein expression by Western blotting. Nuclear proteins were isolated to evaluated the expression of β-catenin, YAP1 and PPARγ proteins through Western blotting, accompanied by a quantitative analysis of band density. GAPDH and Lamin A/C served as the internal controls for cytoplasmic and nuclear extract, respectively. All the histograms represent the relative expression levels of proteins normalized by GAPDH or Lamin A/C. *P<0.05, **P<0.01, ***P<0.001 vs. the group of MEFs without TL1A treatment.