IL-6/STAT3 pathway reinforces iCAFs polarization and glutamine synthesis in fibroblasts. (A) qPCR analysis of relative mRNA expression levels of glutamine synthesis pathway associated genes in NFs treated with TCM at indicated different time points. (B) Western blots of GS protein expression level in NFs treated with TCM at indicated time points. (C) qPCR analysis of relative mRNA expression levels of glutamine synthesis pathway associated genes in NFs treated with TCM or MEF CM for 24 h. (D) Glutamine levels in spent culture medium of NFs treated with TCM or MEF-conditioned medium (MEF CM) for 24 h. Glutamine levels are normalized to unspent medium and shown relative to NFs glutamine production in normal medium (Ctr). (E and F) GSEA for iCAF (E), IL-6/JAK/STAT3 signaling pathway (F) in NFs treated with TCM versus control. Significant NES >1; q value <0.05. (G) Bubble plots comparing expression of genes associated with glutamine synthesis pathway in mCAF and iCAF. (H) Western blots of STAT3 were shown in NFs treated with or without TCM for 24 h. Quantification was calculated by ImageJ. (I) qPCR analysis of relative mRNA expression levels of glutamine synthesis pathway associated genes in NFs treated with Yumm1.7 CM in the presence of anti–IL-6 neutralization antibody (100 μg/ml). (J) ELISA of IL-6 protein level in the supernatant of NFs treated with or without TCM. (K) qPCR analysis of relative mRNA expression of GS in TCM treated NFs after STAT3 knockdown. Data are the cumulative results from at least two independent experiments (D) or are representative of at least two independent experiments with similar results (A–C and H–K). All data are shown as mean ± SEM and were analyzed by two-tailed, unpaired Student’s t test (A, B, H, and J), one-way ANOVA with Tukey’s multiple comparison test (C), or two-way ANOVA with Sidak’s multiple comparison test (I and K). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Source data are available for this figure: SourceData F2.