Figure 4(A) Human fibroblasts (BJ) were treated with vehicle (water for 8 times 24 hours), abemaciclib (1 μM for 8 times 24 hours) or doxorubicin (250 nM for 24 hours). RNA-sequencing was performed with treated cells at 8 dpt (n=3 independent samples, sequenced together) and heatmap of the p53-associated SASP for doxorubicin- or abemaciclib-treated groups (8 dpt) relative to vehicle-treated group from the RNA-sequencing datasets (p-value<0.01 was regarded as significant). (B) Human BJ fibroblasts, hTERT-RPE1 epithelial cells and lung mesenchymal stem cells were treated with vehicle (DMSO for 8 times 24 hours) or doxorubicin (250 nM for 24 hours) or paclitaxel (50 nM for 24 hours) or palbociclib (1 μM for 8 times 24 hours) or abemaciclib (1 μM for 8 times 24 hours). At 8 dpt, RNA was isolated and mRNA for the indicated p53-associated SASP genes quantified by qRT-PCR relative to tubulin (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001. (C) Chromatin was extracted from BJ fibroblasts treated with vehicle or abemaciclib or nutlin-3a and ChIP assays using an antibody against p53 were performed. qRT-PCR was performed using primers amplifying the promoter region of IGFBP3 containing p53 binding sites. Values indicate fold enrichment relative to the vehicle group (n=4 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001. (D and E) RNA was isolated from vehicle, abemaciclib (1 μM) (D) or palbociclib (1 μM) (E) treated scramble/shp53 BJ cells and quantified by qRT-PCR for IGFBP3 or LIF genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001. (F) RNA isolated from kidneys treated with doxorubicin or abemaciclib and mRNA encoding indicated p53-associated SASP genes quantified by qRT-PCR (n=6 mice/group). (G) RNA was isolated from kidneys of p16-3MR mice treated with abemaciclib (50 mg/kg, 7 consecutive days) ± pifithrin-α (2 mg/kg, 7 consecutive days), and mRNA encoding p53 associated SASP genes was quantified by qRT-PCR and normalized on tubulin (n=5 mice/group). ( Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001. (H) Cell-free plasma was derived from breast cancer patients treated with paclitaxel (n=10) or palbociclib (n=9) and the expression levels of IGFBP3 quantified by ELISA. The values represent the ratio between on-treatment and pre-treatment levels for each patient. Data information: Data are means ±SD. Unpaired student t-test *p<0.05, **p<0.01, ***p<0.001. (I) RNA was isolated from vehicle or nutlin-3a (1 μM for 8 times 24 hours) treated BJ cells at 8 dpt and quantified by qRT-PCR for p53 target cell cycle genes, SASP genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001. (J) Mouse dermal fibroblasts (MDF) or MDF-p53-/- fibroblasts were transfected with empty vector (EV) or p53-overexpressing vector (p53OE), then the cells were treated with vehicle (water) or doxorubicin (250 nM for 24 hours) and qRT-PCR was performed for Igfbp3 and Lif genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001. EV, empty vector