Fig. 4

NPM1 mediates DDP sensitivity in OS cells through its phosphorylation at Ser260. a The Efficiency of siRNA-mediated NPM1 knockdown in 143B, HOS, U2OS, and U2OS/DDP was determined by WB. b The IC50 of OS cell from A was calculated after the addition of gradient concentration of DDP for 24 h. P values were assessed by the one-way ANOVA. c, d The OS cells from A were subjected to treatment with DDP for 7–15 days (143B:0.2 μM, HOS and U2OS: 0.4 μM, U2OS/DDP: 1 μM). Cell survival was determined by colony formation assay. e Quantification of clone formation number in NPM1 knockdown cells treated with DDP. Error bars represent ± SD from three independent experiments. f–g Apoptosis analysis of NPM1 knockdown cells treated with DDP for 24 h (143B:1 μM, HOS and U2OS:2 μM, U2OS/DDP: 10 μM). h Quantification of apoptosis rate of NPM1 knockdown cells treated with DDP. Error bars represent ± SD from three independent experiments. i–k NPM1 knockdown 143B cells were subcutaneously injected into nude mice. Starting on day 10, mice were treated with DDP (3.5 mg/kg) by i.p. injection once every 4 days for a total of three injections. i Representative images of tumors in each group after surgical resection. j Tumor volume measured on the indicated day represented as mean ± SD. k Tumor weights were measured and represented as mean tumor weight ± SD. l The NPM1 knockdown OS cells (143B, HOS, U2OS, and U2OS/DDP) were transiently transfected with the NPM1-WT, NPM1-S260A, and NPM1-S260D. Following the gradient concentration of DDP treatment for 24 h, the IC50 value was determined by MTT assay. m The NPM1 knockdown OS cells (143B, HOS, U2OS, and U2OS/DDP) were exogenous expressions of NPM1-WT, NPM1-S260A, and NPM1-S260D, following treatment with DDP for 7–15 days (143B:0.2 μM, HOS and U2OS:0.4 μM, U2OS/DDP: 1 μM). Cell survival was determined by colony formation assay. n The NPM1 knockdown OS cells (U2OS/DDP-Luc) were exogenous expressions of NPM1-WT, NPM1-S260A, and NPM1-S260D, following were orthotopically injected into mice. Starting on day 10, mice were treated with DDP (3.5 mg/kg) by i.p. injection once every 4 days for a total of three injections. (Above) Representative images of living imaging of xenograft mouse model. (Below) Representative immunohistochemical images Ki67 in the indicated xenografts tumors. Data shown are mean ± SD Error bars, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001