Fig 2: Nucleostemin-dependent I3C stimulation of MDM2 nuclear compartmentalization and localization into nucleolus foci. 10AT-Her2 cells were transfected with either control scramble siRNA or nucleostemin siRNA, and then treated with or without 200 μM I3C for 48 hours. (A) The level of nucleostemin protein was determined by Western blot analysis. (B) Cell extracts were biochemically separated into nuclear enriched and cytoplasmic fractions, electrophoretically fractionated, and Western blots probed with antibodies specific for MDM2, the cytoplasmic marker HSP90 and the nuclear marker lamin. (C) The subcellular localization of MDM2 and nucleostemin was determined by indirect immunofluorescence microscopy. DAPI staining was used to visualize DNA stained nuclei. Scale bar represents 4 μm. DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; I3C, indole-3-carbinol; MDM2, murine double mutant 2; NS, nucleostemin; siRNA, small interfering RNA.

Fig 3: Effects of nucleostemin knockdown on I3C-induced apoptotic response and regulated MDM2- p53 protein interactions. (A) 10AT-Her2 cells transfected with nucleostemin siRNA or with scrambled siRNA control were treated with or without 200 µM I3C for the indicated durations. The relative amount of apoptosis was quantified by the ratio of sub-G1 DNA content as determined by flow cytometry. The number of apoptotic cells observed after I3C treatment was normalized to the number of apoptotic cells observed in DMSO-vehicle-control-treated cells. (B) 10AT-Her2 cells were transfected with either scrambled siRNA or nucleostemin-specific siRNA, and then treated with or without 200 µM I3C for 48 hours. Total cell extracts were immunoprecipitated with MDM2 antibodies, electrophoretically fractionated and Western blots probed with p53-specific antibodies. Negative control immunoprecipitations were carried out with non-immune antibodies (IgG) or samples that were not immunoprecipitated (No IP). (C) Western blots of total cell extracts (total protein) were analyzed for p53, serine-166 phosphorylated MDM2 and total MDM2. DMSO, dimethyl sulfoxide; I3C, indole-3-carbinol; IgG, immunoglobulin G; IP, immunoprecipitated; MDM2, murine double mutant 2; NS, nucleostemin; siRNA, small interfering RNA.

Fig 4: E2F1 promotes transcription of SPIN1. (A) Schematic diagram of the putative SPIN1 promoter regions (-2022/0, -1004/0, -508/0, -374/0, -253/0, -150/0, and -75/0). These promoter fragments were PCR-amplified from the genomic DNA of BGC823 cells, which were then inserted into the HindIII-NheI sites upstream of the firefly luciferase in the pGL3-Basic vector. (B) Transcriptional activity analysis of the putative SPIN1 promoter fragments in 293T cells by dual-luciferase reporter assays. Three independent experiments were performed, and data are presented as mean ± SD (unpaired t-test with Welch's correction,*P < 0.05). (C, D) Dual-luciferase reporter assays show that E2F1 and SP1 increase promoter activities of pGL3-374/0. Three independent experiments were performed, and data are presented as mean ± SD (unpaired t-test, **P < 0.01). (E, F) RT-qPCR assay indicates that E2F1 promotes the expression level of SPIN1 in MKN45 cells, whereas SP1 does not. Three independent experiments were performed, and data are presented as mean ± SD (unpaired t-test, **P < 0.01). (G) Western blot assay indicates that E2F1 promotes the expression level of SPIN1, as well as its target genes MDM2, P-RB, while the expression level of p21 is decreased. Three independent experiments were performed. (H) ChIP-PCR assay shows that PCR products were amplified from the DNA fragment which was immunoprecipitated by the anti-E2F1 antibody using primers that covered the four E2F1-binding sites. Three independent experiments were performed. (I, J) Approximate 150-fold (MKN45) and 20-fold (BGC823) enrichment of the promoter amplifications of SPIN1 in the four binding sites in GC cells was observed. Three independent experiments were performed, and data are presented as mean ± SD.

Fig 5: Effects of Nutlin-3 on mutant and wtp53 melanoma cells. (a) Cell lysates were prepared from melanoma cell lines grown in complete media and analyzed by WB for p53, Mdm2 and GAPDH as a loading control. The cell line p53 status is indicated as mtp53 or wtp53. (b) Cell viability of the Nutlin-3-treated cells as indicated, was evaluated by PrestoBlue assay and displayed as a percentage of untreated controls. Data correspond to the mean±s.e.m. from three independent experiments. Statistical analysis: Nutlin-3-treated cells compared with control-treated cells: *P<0.05, ***P<0.001.