Fig 2: 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 3: 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.

Fig 4: The APC substrates SKP2 and Cyclin B exhibit a phosphorylation-dependent mechanism for the interaction with core APC during cell cycle progression.a Western blot of HeLa cells synchronized by double thymidine block using phospho-specific antibodies. b Western blot of immunoprecipitates from HeLa cells synchronized by double thymidine block and collected at serial times for immunoprecipitation using SKP2 (upper panel), Cyclin B (middle panel), or ID2 (lower panel) antibodies. E47 and HEB are controls for the immunoprecipitation of endogenous ID2. c Loss of CDH1-mediated regulation of SKP2-S64D stability. Western blot of HeLa cells expressing FLAG-SKP2 WT, S64A, or S64D in the presence or absence of CDH1. d Loss of CDH1-mediated regulation of Cyclin B-S126D stability. Western blot of HeLa cells expressing FLAG-Cyclin B WT, S126A, or S126D in the presence or absence of CDH1. e In vivo ubiquitylation of HeLa cells co-expressing FLAG-SKP2 WT or phospho-mutants and HA-ubiquitin. HA western blot of cellular lyasates immunoprecipitated with FLAG antibody. WCL, whole cellular lysates. f In vivo ubiquitylation of HeLa cells co-expressing FLAG-Cyclin B1 WT or phospho-mutants and HA-ubiquitin. HA western blot of cellular lysates immunoprecipitated with FLAG antibody. WCL whole cellular lysates. g Loss of interaction between the SKP2-S64D and core APC is independent of CDH1. FLAG-immunoprecipitation-western blot of HeLa cells expressing FLAG-SKP2 WT or phospho-mutants in the presence or the absence of CDH1 siRNA (left panel). Right panel, whole cellular lysate (WCL). h Loss of interaction between the Cyclin B-S126D and core APC is independent of CDH1. FLAG-immunoprecipitation-western blot of HeLa cells expressing FLAG-Cyclin B WT or phospho-mutants in the presence or the absence of CDH1 siRNA (left panel). Right panel, whole cellular lysate (WCL). Total and phosphorylated proteins are from independent gels; APC1, APC3, CDH1 are from the same gel in each panel; E47, HEB, pHH3, SKP2 and Cyclin B1 are from independent gels; HA and FLAG/Vinculin in panel e are from different gels; Loading controls are from the same gel as FLAG or HEB in panel a. Molecular weight markers are indicated in kDa. Experiments were repeated two times with similar results.

Fig 5: 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.