Fig. 4: Cytoprotective effect of NSF-HA depends on p53.

a Western blots showing p-p53-Ser9 levels in IMR90 cells treated with 20 μg/ml cNSF-HA or fNSF-HA for 6 h. b Quantification of Western blots shown in (a) by Image J. Averages are of three biological replicates. For each biological replicate, immunoblotting was performed three times and averaged. c p53-knockdown abrogates the cytoprotective effect of NSF-HA on oxidative stress-induced cell death in IMR90 cells. The percentages of live cells were measured by Annexin-V/PI staining 1 day after 1 h of 3 mM tBHP-treatment (n = 3). Cells were pre-incubated for 6 h with 20 μg/ml NSF-HA or PBS before tBHP-treatment. siRNA was transfected 2 days before the first tBHP-treatment. d p53-knockdown abrogates the cytoprotective effect of NSF-HA on oxidative stress-induced growth arrest in IMR90 cells (n = 4). Cells were pre-incubated for 6 h with 20 μg/ml NSF-HA or PBS for 6 h and then HA was removed and cells were exposed to 200 μM tBHP for 1 h on day 0 and day 1. siRNA was transfected 2 days before day 0. e Confirmation of p53-knockdown in IMR90 cells by Western blot. Cells were collected 2 days after p53 or control siRNA transfection. Immunoblotting was repeated once with similar result. f p53-knockout abrogates the cytoprotective effect of NSF-HA on oxidative stress-induced cell death in MSF. The percentages of live MSF were measured by Annexin-V/PI staining 1 day after 1 h of 1.5 mM tBHP-treatment (n = 3). Cells were pre-incubated for 6 h with 20 μg/ml NSF-HA or PBS before tBHP-treatment. g p53-knockout abrogates the cytoprotective effect of NSF-HA on oxidative stress-induced growth arrest in MSF (n = 4). Cells were pre-incubated for 6 h with 20-μg/ml NSF-HA or PBS for 6 h and then HA was removed and cells were exposed to 150-μM tBHP for 1 h on day 0 and day 1. h Confirmation of p53-knockout in MSF by Western blot. Immunoblotting was repeated once with similar result. Error bars are presented as mean ± SD values. *p < 0.05 (two-tailed t-test).