Fig 2: Subcellular localization of c-Myc and AZ2 (a) Panc-1 cells were transiently transfected with either ECFP-AZ2 or EYFP-c-Myc or both, cultured for 24 h, and observed under a fluorescence microscopy. Immunofluorescence images show the localization of individually expressed ECFP-AZ2 (upper, AZ2 only), EYFP-c-Myc (upper, c-Myc only). Monochrome images of AZ2 and c-Myc were colored in cyan and green, respectively, and images of nuclei stained with Hoechst 33342 were colored in yellow. Nuclear dominant images of AZ2 and c-Myc are indicated. NO, nucleolar dominant distribution, N, nuclear dominant distribution; N/C, nuclear and cytosolic distribution, C, cytosolic dominant distribution. (b) Panc-1 cells were transiently transfected with both ECFP-AZ2 and EYFP-c-Myc. Subcellular localization of the proteins was analyzed as in (a). In the coexpression cells, localization of AZ2 and c-Myc was identical. (c) Panc-1 cells were transiently transfected with either ECFP-AZ2 or EYFP-c-Myc. After 24 h, cells were treated with 20 µM MG132 for 5 h. Then cells were fixed and subcellular localization of the proteins was analyzed as in (a). Nucleolar distribution was confirmed by detecting nucleolar protein, fibrillarin using anti-fibrillarin (Fib) antibody and secondary antibody, AlexaFluor555 (orange)-conjugated anti-rabbit IgG as described in methods. NAgg, nuclear dominant distribution with aggregated forms. (d) Panc-1 cells were transiently transfected with both ECFP-AZ2 and EYFP-c-Myc, and after 24 h, cells were treated with 20 µM MG132 for 5 h. Subcellular distribution of these proteins were analyzed as in (c). (e) Panc-1 cells were transfected with c-Myc siRNA or control siRNA. After 24 h, the cells were transfected with HA-AZ2 (human) and incubated for further 24 h. Then the cells were treated with MG132 for 5 h and immunostained with anti-HA antibody and secondary antibody conjugated with AlexaFluor 555. Monochrome images of AZ2 and nuclei were colored in orange and cyan, respectively. A phase-contrast image was added to confirm the location of nucleoli (siCont + MG). Bar graphs represent quantification of the cells with subcellular localization of the proteins. C, cytosolic distribution. Bar graphs on the right of each images represent quantification of 50 cells (a–d) or 100 cells (e). Data (a–e) shown represent the mean ± SD calculated from three independent experiments. Scale bars, 20 µm.

Fig 3: Interaction between AZ2 and c-Myc. (a) cDNA for HA-c-Myc was transfected with either FLAG-AZ1, FLAG-AZ2 or FLAG-ODC in 293-F cells. Cell lysates were immunoprecipitated with anti-FLAG antibody (IP: FLAG) and the resulting precipitates as well as the original cell lysates (input), were subjected to immunoblot analysis with anti-HA or anti-FLAG antibody. The experiment was repeated three times (b) The above experiment was performed with swapped tags (HA-AZ1, HA-AZ2, HA-ODC and FLAG-c-Myc). Immunoprecipitaion was performed with anti-FLAG antibody and c-Myc bound proteins were detected with anti-HA antibody. Expressed protein levels in cell lysates were checked by immunoblotting with anti-HA antibody. Experiments were repeated three times. (c) In vitro immunoprecipitation assay was performed using Human HA-AZ2, HA-c-Myc or HA-ODC purified from 293-F cells. Purified proteins were mixed in M-PER buffer, and after overnight incubation at 4 °C, HA-c-Myc was immunoprecipitated by anti-c-Myc antibody and c-Myc bound protein was detected by immunoblotting using anti-c-Myc antibody. Detailed protocol for in vitro immunoprecipitation assay indicated in Methods.

Fig 4: A model for AZ2-mediated c-Myc degradation in the nucleolus. Stress stimuli such as hypoxia and glucose deprivation increase cellular polyamine levels, resulting in induction of AZ2. AZ2 shuttles between cytoplasm and nucleus, and interacts with c-Myc in the nucleoplasm. c-Myc takes AZ2 to the nucleolus, where AZ2 accelerates c-Myc degradation by the proteasome without ubiquitination. This pathway is independent of the known pathway that requires Thr-58 phosphorylation and ubiquitination of c-Myc by Fbxw7 (Ubiquitin Pathway).

Fig 5: Reduced autocrine activity of IFNß produces a state of low ISG expression. A, LHS EV and MCF10A EV cells were treated with anti-hIFNß or PBS. Transcript levels of ISGs relative to GUSß reference gene were quantified by qPCR. Values were normalized to PBS treatment. Data represent two or three biological replicates. Statistics were calculated using Student’s t test between PBS and anti-hIFNß treatment. B, LHS EV cells were treated with 50 µM GSK8612 or vehicle (DMSO) and characterized by label-free proteomics. Heatmap compares fold change values for a set of ISGs (with p = 0.05) for (left column) LHS MYC versus LHS EV and (right column) LHS EV + GSK8612 versus LHS EV + DMSO. Heatmap represents two biological replicates. C, isogenic oncogene models were stimulated with 500U/ml hIFNß or PBS. mRNA levels of ISG relative to GUSß reference gene were quantified by qPCR. Bar graphs summarize fold change between PBS and IFNß treatment for each cell line, and report mean and standard deviation of biological duplicates. D, cells expressing MYC, KRAS, and AKT oncogenes were pretreated with 500U/ml hIFNß or PBS and subsequently stimulated with transfection agent alone or complexed with polyI:C. Transcript level of IFNß relative to GUSß reference gene was quantified by qPCR. Extent of IFNß induction was calculated as the fold change in IFNß mRNA between polyI:C treatment and transfection agent alone, and values were normalized to the PBS treatment. Bar graphs represent mean and standard deviation for three biological replicates. Statistics were calculated using Student’s t test between PBS and hIFNß treatments. *p = 0.05, **p = 0.005, ***p = 0.0005. dsRNA, double-stranded RNA; dsDNA, double-stranded DNA; EV, empty vector; ISG, interferon-stimulated gene.