Fig 2: Sensitivity of MCF-7 cells to tamoxifen is regulated by p53 and survivin. a Nogo-B receptor (NgBR) knockdown increases p53 protein level and decreases survivin in MCF-7-TamR cells. Protein levels were determined by western blot analysis. b Quantitative analyses of proteins presented in Fig. 3a were carried out using ImageJ and were normalized to the housekeeping gene ß-actin. Data are presented as fold changes in the siNgBR group compared to the non-silencing (NS) group. c Knockdown of p53 increases survivin level in MCF-7 cells. MCF-7 cells were transfected with siRNA specifically targeting p53 as described in “Methods”. The protein levels of p53, NgBR, survivin and ß-actin were determined using western blot analysis. d Quantitative analyses of proteins presented in Fig. 3c were carried out using ImageJ and were normalized to ß-actin. Data are presented as fold changes in the sip53 group compared to the NS group. e Knockdown of p53 increases the clonogenenicity of MCF-7 cells. Clonogenic survival assay was used for measuring clonogenicity of MCF-7 cells treated with 4-OHT (1 µM). f Quantification of colony number in colony formation assays is presented in Fig. 3e. g Survivin knockdown increases apoptosis of MCF-7-TamR cells induced by 4-OHT (5 µM). h Percentages of apoptotic cells in Fig. 3g are shown in the bar graph. i Knockdown of p53 decreases the sensitivity of NgBR-deficient MCF-7 cells to tamoxifen. NgBR knockdown restored the sensitivity to tamoxifen, which is attenuated by silencing p53. j Quantification of colony number in colony formation assays described in Fig. 3i. The data are from three separate repeat experiments, and are presented as the mean ± SD (*p < 0.05, n = 3)

Fig 3: BACH1 aggravates endothelial cell senescence under oxidative stress. (A) Western blot analysis of P21, P53, and BACH1 protein levels in HUVECs under H2O2 (0, 30, 60, 90, 120 µM) treatment for 12 h. Quantitative analysis was normalized to control (n = 3 independent experiments, data are mean ± SEM. One-way ANOVA followed by Tukey post-hoc tests, *p < 0.05 versus Control, **p < 0.01 versus Control). (B) Representative images of SA-ß-gal staining for HUVECs under H2O2 (60 or 120 µM) treatment for 12 h after BACH1 overexpression or knockdown via adenovirus infection. Scale bar, 50 µm. Quantitative analysis was measured by the percentage of SA-ß-gal positive cells (n = 3 independent experiments, data are mean ± SEM, unpaired 2-tailed t-test, *p < 0.05, **p < 0.01). (C) Western blot analysis of P21 and P53 and BACH1 protein levels in HUVECs under H2O2 treatment for 12 h after BACH1 overexpression or knockdown via adenovirus infection. Quantitative analysis was normalized to the control group (n = 3 independent experiments, data are mean ± SEM, unpaired 2-tailed t-test, *p < 0.05, **p < 0.01). (D) qRT-PCR analysis of MCP-1, ICAM1, and IL-6 mRNA levels in HUVECs under H2O2 treatment for 12 h after BACH1 knockdown via adenovirus infection. Quantitative analysis was normalized to the control group (n = 3 independent experiments, data are mean ± SEM, unpaired 2-tailed t-test, *p < 0.05, **p < 0.01).

Fig 4: p53 deletion promotes aberrantly high NEK2 expression through indirect transcriptional regulation. A,B) Luciferase activity driven by NEK2 promoter with normal (full length) or mutant p53 binding site in HEK293-TP53KO and ARP1 cells transiently co-transfected with p53 OE. Data presented as mean ± SD, p-values are calculated using unpaired t test, n = 3, **p < 0.01. C) ChIP confirming that the transcription factor p53 specifically binds to the NEK2 promoter region in H929 cells. Data presented as mean ± SD, p-values are calculated using unpaired t test, n = 3, ****p < 0.0001. D) Correlation of expression between NEK2 and E2F8 in MM patients based on GEP database (GSE2658, n = 559, p-values are calculated Spearman correlation coefficient ). E) The mRNA and protein levels of TP53, CDKN1A (p21), and E2F8 in H929 cells with or without p53 deletion. Here and in panels (F–J), mRNA and protein data were obtained using qPCR and immunoblotting, respectively. Data presented as mean ± SD, p-values are calculated using unpaired t test, n = 3, ***p < 0.001. F) The mRNA and protein levels of TP53, CDKN1A (p21), and E2F8 in ARP1 cells with or without p53 overexpression. Data presented as mean ± SD, p-values are calculated using unpaired t test, n = 3, *p < 0.05, ***p < 0.001. G,H) The mRNA and protein levels of NEK2 and E2F8 in H929-Ctrl and H929-TP53KO cells with (H929-TP53KO/E2F8 OE) or without E2F8 overexpression. Data presented as mean ± SD, p-values are calculated using unpaired t test, n = 3, *p < 0.05, **p < 0.01. I,J) The mRNA and protein levels of NEK2 and E2F8 in ARP1-EV and ARP1-TP53 OE cells with (ARP1-TP53 OE/E2F8 OE) or without E2F8 overexpression. Data presented as mean ± SD, p-values are calculated using unpaired t test, n = 3, **p < 0.01. K) ChIP confirmed that the transcription factor E2F8 specifically binds to the NEK2 promoter region. L) Schematic of p53 deletion enhancing NEK2 expression through E2F8 upregulation.

Fig 5: IGF-1 gene transfer to CCl4-induced rat models reduces nuclear progerin and p53 protein levels of primary rat hepatocytes.a The H2O2 content of primary rat hepatocytes, isolated from CCl4-induced rat models on Day 6 and Day 28. *P < 0.05 versus the Vehicle group on Day 6; #P < 0.05 versus the CCl4 + LV-CTR group on Day 6; &P < 0.05 versus the Vehicle group on Day 28; $P < 0.05 versus the CCl4 + LV-CTR group on Day 28. b Representative immunoblots of progerin, Lamin A/C, and p53 of primary hepatocytes isolated from the CCl4-induced rat models on Day 6 and Day 28. c The progerin protein expression (red) in hepatocytes of the four groups (Vehicle, CCl4, CCl4 + LV-CTR, CCl4 + LV-IGF-1) on Day 6 and Day 28, shown by immunofluorescence. Nuclear were showed by DAPI (blue). Scale bar: 50 µm. n = 6 per group