Journal: iScience

Article Title: FGL2-induced metabolic dysregulation in enteric neural crest cells provides insight into Hirschsprung disease pathogenesis

doi: 10.1016/j.isci.2025.113423

Figure Lengend Snippet: FGL2 enhances OXPHOS via the JAK2–STAT3 signaling pathway in ENCCs (A) Gene Set Enrichment Analysis (GSEA) of RNA-seq data from ENCCs treated with rFGL2 (2.5 μg/mL, 6 h) revealed the upregulation of the JAK-STAT signaling pathway. (B) Immunofluorescence staining showed the nuclear accumulation of phosphorylated STAT3 (p-STAT3) upon FGL2 stimulation, which was reduced by pretreatment with JAK2 inhibitor AG490 (50 μM) or STAT3 inhibitor Stattic (5 μM). Scale bar = 20 μm. n = 3 wells per group. (C) Western blot analysis of phosphorylated and total JAK2 and STAT3 in ENCCs treated with FGL2, AG490, or Stattic. Quantification confirmed the activation of JAK2–STAT3 by FGL2 and suppression by respective inhibitors. Data are presented as mean ± SEM, n = 3 wells per group. Western blot analysis of representative OXPHOS complex proteins (ATP5A1, UQCRC1, SDHB, MTCO2, NDUFB8) revealed increased expression after FGL2 treatment, attenuated by JAK2 or STAT3 inhibition. β-ACTIN was used as a loading control. n = 3 wells per group. (D) Seahorse XF Cell Mito Stress Test showed increased oxygen consumption rate (OCR) in ENCCs following FGL2 treatment, which was significantly reversed by the inhibition of JAK2 or STAT3. Basal respiration, maximal respiration, spare respiratory capacity, and proton leak were quantified, n = 3 wells per group. (E and F) Flow cytometry detection of intracellular ROS levels (DCFH-DA staining) indicated that FGL2-induced ROS accumulation was reduced upon treatment with AG490 or Stattic. Data are presented as mean ± SEM. Statistical analysis was performed using an unpaired two-tailed t-test. Significance reported as ∗ p < 0.05, ∗∗ p < 0.01. n = 3 wells per group.

Article Snippet: Anti-rabbit Phospho-Jak2 (Tyr1007/1008) Antibody , Cell Signaling Technology , Cat# 3771; RRID: AB_330403.

Techniques: RNA Sequencing, Immunofluorescence, Staining, Western Blot, Activation Assay, Expressing, Inhibition, Control, Flow Cytometry, Two Tailed Test