Fig. 6

The GAS7 gene expression was transcriptionally regulated by p53. a MCF-7 cells were transfected with control or GAS7 siRNA for 48 h, and the protein lysates were analyzed by western blotting using anti-p53, anti-GAS7, and anti-GAPDH antibodies. b ChIP assay was performed to detect p53 putative binding sites on the GAS7 promoter in MCF-7 cells. The 2% of total lysate DNA (input), DNA pulled-down by using histone H3 antibody (H3), p53 antibody (p53), IgG, or no antibody (N) were amplified using primers for putative p53 binding region in the GAS7 promoter. The PCR products were analyzed in 1.5% agarose gel as shown. c Genomic DNA from 339 breast cancer patients (including age ≤ 40 years old, n = 135; age > 40 years old, n = 204) were analyzed for the mutation of p53 gene by Sequenom MassArray platform or whole exon sequencing technology. The χ² test statistics analysis was used (*p < 0.05). d GAS7b mRNA expression was analyzed by RT-qPCR for the breast cancer patients with wild-type (n = 40) or mutant p53 gene (n = 39). The Student’s t-test was used to compare these two groups (*p < 0.05). e Upper: schematic diagram illustrating the protein domains of p53 gene, and four mutation hotspots as indicated were found in our analysis. Bottom: dual-luciferase reporter assay showing MCF-7 cells co-transfected with PGL4-GAS7 promoter and PGL4-Renilla luciferase plasmid as internal control, and subsequently transfected with empty vector, wild-type p53, R175H, G244D, R248Q, or R342stop of p53 mutant expressing plasmid. Histograms represent normalized mean ± SD (n = 3). f Western blotting was performed to detect the expression of GAS7 and p21 under the regulations by wild-type or mutant p53 in MCF-7 cells. GAPDH protein was served as the loading control