Fig 2: Screening of hub SE-associated gene IGF2BP2 in HNSCC. A The ROSE algorithm was utilized to plot the enhancer regions of normal oropharyngeal mucosa and HNSCC cell lines CAL27, Detroit562, HN12, and SCC25 in increasing order based on the H3K27Ac ChIP-seq signal, and enhancers above the inflection point of curve are defined as SEs. B In a comprehensive comparison of these HNSCC cell lines, 771 specific SE-associated genes and 442 specific SE-associated protein-encoding genes (mRNA) were identified, compared to SE-associated genes in normal oropharyngeal mucosa. C Disease-gene network enrichment analysis revealed that HNSCC cell-specific SE-associated genes were most enriched in carcinoma of the larynx. D-F WGCNA analysis identified the most relevant modules in the TCGA-HNSCC (D), GSE30784 (E), and GSE42743 (F) datasets for HNSCC, which were represented by the green (r = 0.47), turquoise (r = 0.74), and black (r = 0.71) modules, respectively. G Wayne diagram depicted the intersection of HNSCC cell-specific SE-associated protein-encoding genes with HNSCC-associated genes obtained from TCGA-HNSCC, GSE30784, and GSE42743 in WGCNA

Fig 3: SE-associated IGF2BP2 is transcriptionally activated by KLF7. A Wayne diagram depicted the intersection of transcription factors bound to IGF2BP2-SE and promoter. B-E Correlation of IGF2BP2 expression with KLF7 in four HNSCC datasets, TCGA-HNSCC (B), GSE30784 (C), GSE42743 (D), and GSE41613 (E). F–H TCGA-HNSCC (F), GSE30784 (G), and GSE42743 (H) datasets revealed that KLF7 expression was upregulated in HNSCC. I and J Poor overall survival in patients with high KLF7 expression in the TCGA-HSCCC (I) and GSE42743 (J) datasets. K Western blot was used to assess the protein level of IGF2BP2 after silencing KLF7. L and M QRT-PCR detected the relative IGF2BP2 expression (M) after silencing KLF7 (L). N Western blot was performed to detect the protein level of IGF2BP2 after stable overexpression of KLF7. O and P QRT-PCR were used to assess the relative IGF2BP2 expression (P) after stable overexpression of KLF7 (O). Q Prediction of KLF7 binding sites and binding motifs in IGF2BP2-SE and promoter regions using the JASPAR website. R Binding sites and binding motifs of KLF7 in the SE region of the IGF2BP2 coding chain. S Schematic representation of wild-type and mutant KLF7 binding motifs in IGF2BP2-SE region. T Reduced luciferase activity after mutating the binding site of KLF7 to IGF2BP2-SE region. U The bindings of KLF7 to the IGF2BP2-SE and promoter regions were examined by ChIP-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001. All the data are presented as mean ± SD from three independently performed experiments

Fig 4: IGF2BP2 promotes pancreatic cancer cell proliferation.a Ectopic expression of IGF2BP2 promotes cell proliferation in BXPC-3 cells as detected by MTT assays. b IGF2BP2 promote colony formation in BXPC-3 cells. c Suppression of IGF2BP2 by IGF2BP2-siRNAs (siRNA#1, siRNA#2, siRNA#3) as determined by western blot. Top, BXPC-3; bottom, SW1990. d In contrast to ectopic expression, IGF2BP2 siRNAs significantly suppresses cell growth in BXPC-3 and SW1990 cells. e IGF2BP2 silencing significantly reduces colony formation in BXPC-3 and SW1990 cells. Values in a, b, d, e are mean ± SEM. **P < 0.01; ***P < 0.001.

Fig 5: Th1- and Th17-extrinsic activities of IMP2.(A) IMP2 expression by Western blot on whole cell lysates from CD4+ T cells cultured in Th0 conditions (lanes 1, 3, 5, 7) and Th17 conditions (IL-6, IL-23, TGF-β, and IL-1β) (lanes 2, 4, 6, 8). Control lysates from Imp2+/+ and Imp2–/– MEFs (lanes 9 and 10). (B) Diagram of in vitro CD4+ T cell polarization conditions. (C–E) CD4+ T cells were isolated from spleen and stimulated for 3 days with plate-bound anti-CD3 and anti-CD28 under Th0 or optimal Th17 conditions (IL-6, IL-23, TGF-β, and IL-1β) for 3 days. (C) Cells were stimulated with PMA/ionomycin for 4 hours, stained for CD4 and IL-17, and quantified by flow cytometry. Right: representative FACS plots from 3 independent experiments. (D) ELISA was performed on supernatants collected after 3 days of treatment with anti-CD3 and anti-CD28. Data were pooled from 2 independent experiments. (E) qPCR of the indicated mRNAs in T cells after 3 days of anti-CD3 and anti-CD28. Data show fold change mean ± SEM from 2 independent experiments. (F) CD4+ T cells were isolated from spleen and stimulated for 3 days with plate-bound anti-CD3 and anti-CD28 under Th0 or Th1 conditions (IL-12). ELISA was performed on supernatants collected after 3 days of anti-CD3 plus anti-CD28. (G and H) CD4+ T cells were isolated from spleen and stimulated for 3 days with plate-bound anti-CD3 and anti-CD28 under Th0 or optimal Th17 conditions (G) (IL-6, IL-23, TGF-β, and IL-1β) or suboptimal Th17 conditions (H) (IL-6 and TGF-β) ± CCL2 for 3 days. ELISA was performed on supernatants collected after 3 days of anti-CD3 plus anti-CD28. Each symbol represents 1 mouse. *P < 0.05, **P < 0.01, ****P < 0.0001 by ANOVA with post hoc Tukey’s test.