Fig 2: Ectopic SBDS suppresses tumor growth in vivo.a Ectopic SBDS does not affect body weights of the mice bearing tumors derived from HCT116p53+/+ cells. b Ectopic SBDS suppresses growth of the xenograft tumors derived from HCT116p53+/+ cells in vivo. c Representation of the dissected tumors derived from HCT116p53+/+ cells expressing the empty vector or ectopic SBDS. d, e Ectopic SBDS activates p53 pathway in tumors derived from HCT116p53+/+ cells. The tumors were homogenized and lysed for qRT-PCR d or IB analysis e. f Ectopic SBDS does not affect body weights of the mice bearing tumors derived from HCT116p53-/- cells. g Ectopic SBDS does not affect growth of the xenograft tumors derived from HCT116p53-/- cells in vivo. h Representation of the dissected tumors derived from HCT116p53-/- cells expressing the empty vector or ectopic SBDS. i, j Ectopic SBDS does not affect p53 target gene expression in tumors derived from HCT116p53-/- cells. The tumors were homogenized and lysed for qRT-PCR i or IB analysis j. k Working model of dual regulation of p53 activity by SBDS. Under the normal culture condition, SBDS mostly reside in the nucleolus and cytoplasm to conduct oncogenic function by boosting ribosome biogenesis and maintaining p53 at a low expression level (upper panel). In response to ribosomal stress, SBDS translocates to the nucleoplasm to execute a tumor-suppressive role by interacting with p53 and dissociating the MDM2–p53 complex (lower panel).

Fig 3: Ablation of endogenous SBDS induces RPL5- and RPL11-dependent p53 stabilization.a, b Knockdown of SBDS prolongs p53 protein half-life. HCT116p53+/+ cells were transfected with control or SBDS siRNA for 48–72 hr. CHX was supplemented into the medium for the indicated time before the cells were harvested for IB analysis a. Quantification of the p53/ß-actin ratios is shown in the b. c RPL5 and RPL11 are required for SBDS depletion-induced p53 activation. HCT116p53+/+ cells were transfected with the indicated siRNAs for 48–72 hr and harvested for IB analysis using the antibodies as indicated. d Knockdown of SBDS enhances the RPL11-MDM2 interaction. HCT116p53+/+ cells were transfected with control or SBDS siRNA for ~48 hr. The proteasome inhibitor MG132 was supplemented into the medium for 4 hr before the cells were harvested for co-IP-IB assays using antibodies as indicated. e Knockdown of SBDS enhances the RPL5-MDM2 interaction. The same experiments were conducted as described in d, except that the anti-RPL5 antibody was used.

Fig 4: Ectopic SBDS induces p53 stabilization and activation.a Ectopic expression of SBDS induces the expression of p53 and its target gene p21 in H460 cells. Cells were transfected with the empty vector or SBDS plasmid followed by IB analysis. b Ectopic SBDS induces the expression of p53 and its target gene p21 in a dose-dependent manner in HCT116p53+/+ cells. Cells were transfected with increased dosage of SBDS plasmid and followed by IB analysis. c Lentivirus-delivered ectopic SBDS induces the expression of p53 and its target genes, MDM2 and p21. HCT116p53+/+ cells were infected with control or SBDS-expressing lentiviruses followed by IB analysis. d, e Ectopic SBDS prolongs p53 protein half-life. HCT116p53+/+ cells were transfected with the empty vector or SBDS plasmid for 30–48 hr and treated with CHX for the indicated time followed by IB analysis. The ratios of p53 to GAPDH were presented in e. f Ectopic SBDS counteracts MDM2-induced p53 degradation. HCT116p53+/+ cells were transfected with combinations of Flag-SBDS, HA-MDM2, and empty vector followed by IB analysis. g Ectopic SBDS impairs MDM2-induced p53 ubiquitination. H1299 cells were transfected with combinations of p53, His-Ub, HA-MDM2, and Flag-SBDS followed by in vivo ubiquitination assay and IB analysis. Poly-ubiquitination of p53 was indicated by the open curly brace.

Fig 5: Ectopic SBDS suppresses cancer cell growth in vitro.a, b SBDS promotes apoptosis more dramatically in HCT116p53+/+ cells than that in HCT116p53-/- cells. Cells were transfected with control or SBDS plasmid followed by the Annexin V-FITC and flow cytometry analysis. Quantification of the apoptotic cells are shown in b. c, d SBDS more dramatically suppresses colony-forming ability of HCT116p53+/+ cells than that of HCT116p53-/- cells. Cells were transfected with control or SBDS plasmid followed by the colony formation assay. e, f SBDS inhibits proliferation of HCT116p53+/+ e and H460 cells f. Cells were transfected with control or SBDS plasmid followed by the cell viability assay.