Fig. 4
IL-4 is targeted by TBX21 in A549 cells to maintain cancer stemness. a Functional enrichment map for RNA-based expression revealing TBX21 modulated networks. Nodes (circles) with distinct color subparts represent different function enriched in gene sets from DAVID database. Nodes including distinct color subparts have significant overlap with TBX21 predicted targets, and each node connected with TBX21 belongs to the same cluster (MCL cluster algorithm). Distinct color lines between nodes correspond to different interactions among these significant genes (Wilcoxon test P < 0.05). b Correlation of TBX21 and IL-4 mRNA levels (normalized) in integrated dataset. Spearman’s correlation and P value indicated. c qPCR analysis of TBX21 and IL-4 expression at mRNA level. d Western blot analysis of IL-4 expression with TXB21 knockdown in A549 cells. β-ACTIN is loading control. e Western blot analysis of IL-4 expression with TXB21 ectopic expression. β-ACTIN is loading control. f Western blot analysis showed IL-4 deficiency rescued SOX2 and OCT4 expression in A549-TBX21 cells. β-ACTIN is loading control. g Statistical results of percentage of SP cells with IL-4 knockdown in A549-TBX21 cells. h Statistical results of tumor spheres with IL-4 knockdown in A549-TBX21 cells. i CHIP analysis of TBX21-binding sites on IL-4 gene promoter. j Dual-luciferase assay to detect IL-4 promoter activity after TBX21 overexpression. k Summary of prognostic model associated with LUAD. Schematic includes two lungs with LUAD, microarray screening, predictive model and mechanism investigation. Blue cell in left lung represents LUAD lesion. Heatmap indicates significant genes correlated with prognosis. ***P < 0.0001, **P < 0.01, t-test method. Ellipse demonstrates signaling pathway of TBX21–IL-4 in cancer cells. N/A not available, IL interleukin, sc control shRNA, TSS transcription start site, LUAD lung adenocarcinoma, MCS multiple cloning site, PC positive control, NC negative control