Fig. 3

13q-Enh regulated the phenotypes of Beas-2B cells by enhancing the expression of TNFRSF19. a The TNFRSF19 expression levels were significantly reduced in 13q-Enh^−/− C5 and C23 clones compared with the wild-type Beas-2B cells. (n = 3 per group; error bars are SD; **p < 0.01, unpaired Student’s t test). b A schematic diagram showing the relative positions of the TNFRSF19 gene promoter, 13q-Enh enhancer, AseI restriction enzyme cut sites, and PCR primers for 3C assays (upper panel). 3C assays detected the physical interaction between the TNFRSF19 gene promoter and 13q-Enh element in wild-type Beas-2B cells, but not in 13q-Enh^−/− clones (a mixture of C5 and C23), either using paired PCR primers, A+C or B+D (low panel). c Soft agar assays were performed to determine the effect of restoration of TNFRSF19 expression on NNK-induced malignant transformation in the 13q-Enh^−/− clones. The C5-NNK and C23-NNK clones were infected with the lentivirus vectors expressing TNFRSF19 or the empty and were subsequently incubated in soft agar for colony formation. d The colony numbers were accounted and statistically analyzed. The results showed that restoration of the TNFRSF19 expression significantly reduced the NNK-induced cell transformation. (n = 3 per group; error bars are SD; **p < 0.01, unpaired Student’s t test). e γ-H2AX was used to evaluate the effects of restoration of TNFRSF19 on the NNK-induced DNA damage in C5-NNK and C23-NNK cells. Cell nuclei were stained with Hoechst 33258 (blue), and breaking DNA ends were detected by γ-H2AX antibody (red). f Statistical analysis of the data shown in e indicated that restoration of TNFRSF19 expression significantly suppressed NNK-induced DNA damage. (n = 3 per group; error bars are SD; **p < 0.01, unpaired Student’s t test)