Fig. 3

TBX21 associated with cancer stemness as well as CSC biomarkers. a Heatmap shows significantly high correlation between TBX21 and CSC biomarkers (SOX2, NANOG, KLF4, ALDH1A1 and OCT4), Spearman’s r ≥ 0.85. b Genetic alterations analysis of TBX21 and five CSC biomarkers in LUAD patients (n = 520) with DFS or OS status from TCGA including amplification, deep deletion, mRNA upregulation, truncating mutation and missense mutation. Synthetically altered ratios 6%, 3%, 7%, 11%, 4% and 4% for TBX21, NANOG, OCT4, SOX2, KLF4 and ALDH1A1 in LUAD patients, respectively. c Western blot analysis of SOX2 and OCT4 expression with TBX21 knockdown in A549 cells. β-ACTIN is loading control. d Western blot analysis of TBX21 expression in non-SP or SP cells. β-ACTIN is loading control. e Western blot analysis of SOX2 and OCT4 expression with TXB21 ectopic expression. β-ACTIN is loading control. f Side population assay to detect SP cell proportion with silencing of TBX21. g Side population assay to detect SP cell proportion with TXB21 ectopic expression. h Representative images of tumor spheres with TBX21 deficiency. i Representative images of tumor spheres with TBX21 overexpression. j Statistical results of percentage of SP cells with TXB21 knockdown. k Statistical results of tumor spheres with TXB21 knockdown. l Statistical results of percentage of SP cells with TXB21 overexpression. m qPCR analysis of OCT4 expression at mRNA level in sphere or nonsphere cells. n qPCR analysis of TBX21 expression at mRNA level in sphere or nonsphere cells. o Statistical results of tumor spheres with TXB21 overexpression. p Representative images of tumor burden 12 days after delivery of A549-shTBX21 or A549-sc cells into NOD/SCID mice. q Side population assay to detect SP cell proportion in suspension derived from A549-shTBX21 or A549-sc tumors. r Statistical results of percentage of SP cells between A549-shTBX21 and A549-sc tumors. s Western blot analysis of TXB21, SOX2 and OCT4 expression between A549-shTBX21 and A549-sc tumors. β-ACTIN is loading control. ***P < 0.0001, **P < 0.01, t-test method. LUAD lung adenocarcinoma, TCGA The Cancer Genome Atlas, DFS disease-free survival, OS overall survival, N/A not available, sc control shRNA, SP side population, MCS multiple cloning site