Fig 2: Hyperoxia-induced senescence is p53-dependent and pRB-independent. (a) Cas9 knock-in, p53KO, and pRBKO shown by Western blot, and calnexin (CNX) as a housekeeping control. (b) Senescence levels using C12FDG FACS of WT, pRBKO, and p53KO MLE-12 cells exposed to 21% O2/5% CO2 (Air, white) or 95% O2/5% CO2 (O2, black) for 24 h. (c) Emission spectrum of senescent cells is shown in blue for Air, and O2 is in red. (d) ?H2AX and (e) 53BP1 levels were measured using immunofluorescence. Blue = DAPI, nuclei; Green = protein of interest, Cyan = overlapping blue and green signal. (f) ?H2AX protein levels were measured by Western blot in p53KO and WT cells exposed to hyperoxia. (g) Transcriptional levels of p21, pRB, E2F1, p16, MMP3, MMP10 measured by RT-qPCR. *p < 0.05. MLE-12, mouse lung epithelial

Fig 3: Different protein levels of phosphoserine-18-TRP53, phosphoserine-389-TRP53 and activated-p38MAPK after priming low dose X-ray irradiation in thymocytes of C57BL/6 male and female mice. Left panels, representative Western blots. HIGH indicates exposure to 1.75 Gy of ?-rays. RAR represents radio-adaptive response. LOW denotes exposure to 0.075 Gy of X-rays. Right panels, quantitative data showing normalized relative protein amounts. Each column represents mean of three independent experiments; bars, SD. Open histograms, non-treated thymocytes. Light gray histograms, thymocytes exposed to high dose (1.75 Gy) ?-irradiation. Dark gray histograms, thymocytes exposed to the adaptive regimen of radiation. Black histograms, thymocytes treated with low dose X-irradiation (0.075 Gy). a-b, TRP53. c-d Phosphorylated form of TRP53 at serine 18. e-f Phosphoserine-389-TRP53. g-h Phospho-(Thr180/Tyr182)-p38MAPK

Fig 4: Hyperoxia causes DNA damage and p53 activation. Mouse lung epithelial (MLE-12) cells were exposed to 21% O2/5% CO2 (Air) or 95% O2/5% CO2 (O2) for 24 h. For immunofluorescence, Blue = DAPI, nuclei; Green = protein of interest, Cyan = overlapping blue and green signal. (a) Measured ?H2AX levels using immunofluorescence. (b) Measured 53BP1 levels using immunofluorescence, quantified by macros script. (c) Transcriptional levels of p53, pRB, p16, and p21 measured by RT-qPCR. (d, e) p53 and pRB protein levels measured by western blot. Western blot normalized using calnexin (CNX) levels. (f) p53 nuclear and cytoplasmic levels measured by western blot from nuclear and cytoplasmic extracts. (g) Nuclear quantification of p53 using immunofluorescence. (h) Levels of phosphorylated p53 at Ser15 as measured by ELISA. Sample turns blue according to phosphorylated levels of p53 at Ser15, absorbance measured at 450 nm. *p < 0.05 versus air groups. RT-qPCR, quantitative real time PCR

Fig 5: p53 binds to polymeric actin in the cytoplasm. A. U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h and followed with ETO (10 µM) treatment for another 12 h. The cells were then harvested and subjected to immunoprecipitations using anti-p53 antibody and analyzed by western blotting (a). Western blottings of immunoprecipitated actin were analyzed using Image J software. Results are presented as means ± SD of values from three independent experiments (b). B. U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h and treated with or without ETO (10 µM) for another 12 h. The cells were then harvested and the whole cell extracts were incubated with GST or recombinant GST-p53. The bound proteins were analyzed by western blotting with anti-actin antibody (top panel). GST and GST–p53 were stained with Coomassie Blue (middle panel). Arrows show the position of GST and GST–p53. Whole cell extracts were immunoblotted with antibody against GAPDH to confirm equal loading (bottom panel) (a). Western blottings of actin were analyzed using Image J software. Results are presented as means ± SD of values from three independent experiments (b).C.U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h before YFP-p53 transfection. Twenty-four hours after YFP-p53 transfection, cells were treated with ETO (10 µM) for another 12 h or untreated as control and analyzed by confocalmicroscopy (a). Scale bar, 10 µm. The colocalization ratio of F-actin and YFP-p53 of more than 100 cells were calculated using Image J software. Results are presented as means ± SD of values from three independent experiments (b). All Statistical differences were determined by One-way ANOVA. *, P<0.05; **, P<0.01.