Fig. 5: DSBs promote SPINDOC transcription by KLF4.

a Schematic showing the optimal KLF-binding motif from JASPAR website and the position of this KLF4-binding site in human SPINDOC promoter. b Hela cells were treated with 10-Gy IR and 40μM Etoposide for one hour before media replacement, and then incubated for the indicated timepoints and analyzed for anti-KLF4 expression by Western blot. The data are representatives of three independent experiments. c Hela cells were transfected with siNC or siKLF4 for 48 h and then treated with 40μM Etoposide or DMSO for one hour before media replacement, and then subjected to a further 3 h of recovery. Total RNAs were extracted for RT-qPCR to evaluate SPINDOC mRNA-level changes. Statistical analysis was performed using one-tailed Student’s t-test. Error bas represent mean ± SD, n = 3 biologically independent experiments. *P = 0.0164, ** P = 0.0026, ***P < 0.001. d Hela cells were treated using the same procedure as in (c) and analyzed for SPINDOC and KLF4 protein levels by Western blot. e HEK293T cells were transfected with the indicated plasmids. pGL3-SPINDOC-P represents the cloned 1000-bp SPINDOC promoter depicted in (a). pGL3-SPINDOC-P-Mut harbors a deletion in the KLF4-binding site. At 48 h post-transfection, cells were treated with 5-Gy IR (as indicated) and then allowed 3 h for recovery. Cells were lysed and subjected to a dual-luciferase Firefly/Renilla assay. Error bas represent mean ± SD, n = 3 biologically independent experiments. Statistical analysis was performed using two-tailed Student’s t-test. From left to right representing group 1–8, 3 vs 1, P = 0.0017; 3 vs 2, P = 0.0019; 4 vs 3, 0.0189; 5 vs 3, P = 0.0008; 6 vs 5, P = 0.0040; 7 vs 8, P = 0.0005; 7 vs 6, P = 0.6733; 8 vs 5, P = 0.0016. f Confluent 10-cm-dish HEK293T cells were treated with 10-Gy IR, and allowed to recover for 3 h. Cells were then subjected to ChIP-qPCR analysis using anti-KLF4 and anti-IgG antibodies. % Input was used to depict the relative KLF4 enrichment at the SPINDOC promoter. Graphs represent mean ± SD, n = 3 biologically independent samples. Statistical analysis was performed using two-tailed Student’s t-test with KLF4 vs IgG, P = 0.0010; IR-KLF4 vs IR-IgG, P = 0.0001; NT-KLF4 vs IR-KLF4, P = 0.0090.