Fig 2: Impaired destabilization of the phospho-mimetic ID2 S5D mutant delays mitotic exit.a HeLa cells expressing ID2 WT or S5 phospho-mutants were exposed to double thymidine block and analyzed by flow cytometry. Bar graph shows quantification of cells in G1, S, and G2/M in one representative experiment. Experiments were performed three times with similar results. b HeLa cells treated as in a were immunostained with pHH3 and analyzed by flow cytometry. Bar graph shows quantification of pHH3-positive cells. c Loss of regulation of ID2-S5D during S-G2/M transition. Western blot of HeLa cells expressing FLAG-ID2 WT or S5 phospho-mutants exposed to double thymidine block. d Exit from mitosis is delayed by expression of ID2-S5D mutant. Western blot of HeLa cells expressing FLAG-ID2 WT or ID2-S5D treated with RO3306. e Left panel, schematics of the cell synchronization protocol for the analysis of ubiquitylation of ID2 WT and phospho-mutants at the G2/M and M/G1 transition. Middle panel, western blot of HeLa cells co-expressing FLAG-ID2 WT or phospho-mutants and HA-ubiquitin immunoprecipitated with FLAG antibody. Right panel, western blot of whole cellular lysates (WCL). Molecular weight markers are indicated in kDa. Experiments were repeated two times with similar results. f HeLa cells expressing ID2 wild type or S5 phospho-mutants were transiently transfected with a plasmid expressing the E-box-luciferase cassette. Cells were synchronized by double thymidine block and analyzed for luciferase expression. Bar graph indicates means ± SD of three replicates. p-values are from unpaired t-test, unequal variance. Experiment was repeated three times with similar results. g Loss of binding to chromatin of E47 and HEB in cells overexpressing ID2 S5D. Western blot of chromatin fractions of HeLa cells transfected with ID2 WT or ID2-S5D, synchronized by double thymidine block, and collected at serial times after release. Experiments were repeated two times with similar results. In d, APC3 and Cyclin B1 are from the same blots; pHH3, CDH1 are from independent gels; in e, HA and FLAG are from different gels; in g, E47 and HEB/pHH3 are from different gel. Loading controls are from the same gel as FLAG except in e where α-tubulin is from the same gel as pHH3.

Fig 3: The N-terminal region of ID2 including Ser-5 is necessary for the interaction between ID2 and core APC subunits.a GST-ID2 WT and deletion mutants of the N- and C-terminus were incubated with lysates from HeLa cells. Bound proteins were analyzed by western blot. Lower panel, Coomassie staining of GST-ID2 proteins used in the GST pull-down. b GST pull-down using GST-ID2 WT and N-terminus deletion mutants and HeLa cell lysates. Bound proteins were analyzed by western blot. Lower panel, Coomassie staining of GST-ID2 proteins used in the GST pull-down. c GST pull-down using GST-ID2 proteins and U-2 OS cell lysates followed by western blot. Arrowhead, specific band; asterisk, non-specific band. Lower panel, Coomassie staining of GST-ID2 proteins used in the GST pull-down. d In vivo interaction between ID family members expressed in HeLa cells and core APC subunits, CDH1 coactivator, and bHLH transcription factors (E47 or HEB). ID interacting proteins were detected by FLAG immunoprecipitation followed by western blot for endogenous proteins as indicated. e Interaction between ID2 phosphorylation mutants and D-Box mutant (DBM) expressed in HeLa cells and core APC subunits or CDH1 coactivator. ID2 interacting proteins were detected by FLAG immunoprecipitation followed by western blot for endogenous proteins as indicated. f FLAG-ID2-S5 phospho-mutants expressed in HeLa cells were immunoprecipitated and probed by western blot for the association with endogenous E47. g HeLa cells transduced with non-targeting or CDH1 siRNA were transfected with FLAG-ID2 WT or ID2-S5 phospho-mutants. Cellular lysates were used in FLAG immunoprecipitation followed by western blot for core APC subunits and CDH1 (left panel). Right panel, whole cellular lysates. Molecular weight markers are indicated in kDa. Coomassie staining in a–c was performed on SDS-gels loaded with the same GST-fusion protein amounts used in the binding reactions. APC proteins and CDH1 are from the same blots in each panel; E47 and HEB in panel d are from two independent gels; FLAG is from an independent gel; Loading controls are from the same gel as HEB in d, FLAG in e, E47 in f, APC1/APC3/CDH1 in g. Experiments were repeated two times with similar results.

Fig 4: miR-204 directly inhibits TCF12 expression by targeting its 3′UTR. (A) Predicted binding sites of miR-204 in the 3′UTR of TCF12. Mutant-type was generated in the seed region of the 3′UTR of TCF12 by mutating 3 nucleotides that is recognized by miR-204. (B) Luciferase activity assay was performed in HeLa and C33A cells co-transfected with miR-204 mimics and a luciferase reporter containing the WT Mut 3′UTR of TCF12. (C) The expression of TCF12 in HeLa and C33A cells after 48 h transfection with miR-204 mimics and NC-mim was measured by western blot analysis. **P<0.01. TCF12, transcription factor 12; miR-204, microRNA-204′ 3′UTR, 3′untranslated region; mim-NC, mimic negative control; Mut, mutant; WT, wild-type.

Fig 5: sTWEAK regulates oncomiR-221-3p expression and down-regulates NLK and TCF12 targets. (a) oncomiR-221-3p expression in PC-3 and LNCaP cell extracts and in extracellular vesicles (EVs) isolated from cell media. Different lettering over boxes indicates statistical differences. Significant differences are established at p < 0.05. Data are expressed as mean ± SEM (n = 4 experiments). (b) Selected targets for oncomiR-221-3p by 3 different target prediction algorithms. # conserved elements in multiply-aligned sequences. (c) qRT-PCR mRNA expression of selected oncomiR-221-3p targets in PC-3 cells before and after treatment with sTWEAK. Significant differences: ** p < 0.01; *** p < 0.001. Data are expressed as mean ± SEM (n = 6 experiments). Transcription factor 12 (TCF12); synaptosome associated protein 23 (SNAP23); dipeptidyl peptidase 8 (DPP8); aryl hydrocarbon receptor nuclear translocator (ARNT); Nemo-like kinase (NLK); ZFP36 ring finger protein-like 2 (ZFP36L2); folliculin interacting protein 2 (FNIP2); estrogen receptor 1 (ESR1); homeodomain interacting protein kinase 1 (HIPK1); and scavenger receptor class B-ember 2 (SCARB2). (d) Expression of TCF12, NLK and Fn14 protein in PC-3 cells transfected with the oncomiR-221-3p inhibitor and further treated with sTWEAK. Representative Western blots are presented (top). The membranes were tested with the corresponding antibody. iNC: inhibitor negative control, i221-3p: inhibitor miR-221-3p; Nemo-like kinase (NLK); transcription factor 12 (TCF12); fibroblast growth factor 14 (Fn14). Relative protein expression levels are shown (bottom), which were normalized to the corresponding control β-actin. Different lettering over boxes indicates statistical differences. Significant differences are established at p < 0.05. Data are expressed as mean ± SEM (n = 3 experiments). Full-length blots and gels are presented in Figure S4.