Fig. 3

A SA-β-Gal staining of HCT116 and p53-null HCT116 cells treated with 5 μM e2-I or scrambled e2-S for 96 h (upper panel). And quantitative analysis percentages of SA-β-Gal positive cells (right panel n = 3). Followed by WB with indicated antibodies (lower panel). Scale bars, 25 μm. B Heat map showed the differential expression genes associated with senescence in HCT116 and p53-null HCT116 cells, respectively. Differential expression genes (DEGs) were identified upon e2-I treatment by RNA-Seq. C, D e2-I inhibits mRNA expression of TOPBP1 or TRX1 depending on p53. HCT116 or HCT116-p53 null cells were treated with 5 μM e2-I or scrambled e2-S for 24 h. Total RNA was extracted and real-time quantification reverse transcriptase polymerase chain reaction (QRT-PCR) was performed. E Mutant p53-6A suppresses the expression of TOPBP1. Vectors expressing FLAG tagged p53, p53-6A or p53-6D, were mock-transfected or transfected into p53-null HCT116 cells for 48 h. Then, the nuclear fraction was separated from the cytoplasmic fraction, followed by WB. F SA-β-Gal staining of p53-null HCT116 cells expressing FLAG tagged (p53-WT, p53-6A, p53-6D, and mock) for 96 h (left panel), and quantitative analysis of the percentages of SA-β-Gal positive cells (n = 3, right panel). Scale bars, 25 μm. G Expression of nanobody Nb-28A1 inhibits the phosphorylation of RBM38-Ser195 and p53-Ser315. Vector pcDNA3-HA-Nb-28A1 or control vector pcDNA3-HA-Nb-BV025 was transfected into HCT116 cells for 48 h, followed by WB with indicated antibodies. H Nanobody Nb-28A1 induced cell senescence depending on p53. SA-β-Gal staining of HCT116 or p53-null HCT116 cells expressing HA-Nb-28A1 or HA-Nb-BV025 for 96 h (left panel), and quantitative analysis percentages of SA-β-Gal positive cells (n = 3, right panel). Scale bars, 25 μm. I Expression of nanobody HA-Nb-28A1 for 24 h downregulates the expression of TOPBP1, TRX1, BCL2 or BAD depending on p53. Vector pcDNA3-HA-Nb-28A1 or control vector pcDNA3-HA-Nb-BV025 was transfected into HCT116 or p53-null HCT116 cells for 24 h, followed by WB with indicated antibodies.