-
TRIM21 is involved in the tumorigenesis of several types of tumors[12, 13, 19, 20]. In the present study, we found overexpression of HA-TRIM21 significantly increased U2-OS and Saos-2 osteosarcoma cell viability using MTT assays (Figure 1A-B). Meanwhile, overexpression of HA-TRIM21 protected U2-OS and Saos-2 cells from cytotoxicity in response to several stresses, including exposure to CDDP (30 μg/mL), TRAIL (50 ng/mL), and serum starvation for 24 hours (Figure 1A-B). By contrast, TRIM21 knockdown with siRNA significantly decreased U2-OS and Saos-2 cell viability (Figure 1C-D), as well as amplified cytotoxicity from the aforementioned stresses (Figure 1C-D). These results suggest TRIM21 was a positive regulator of U2-OS and Saos-2 cell proliferation and increased U2-OS and Saos-2 cell tolerance to different stresses.
Figure 1. The effects of TRIM21 on osteosarcoma cell proliferation. (A) U2-OS and (B) Saos-2 cells were transfected with HA-TRIM21 or HA-vector plasmid in 6-well plates and incubated overnight. The cells were then resuspended in McCoy's 5A medium containing fetal bovine serum and seeded into 96-well plates. The cells were then treated with CDDP (30 μg/mL), TRAIL (50 ng/mL), or serum starvation for 24 hours as indicated. MTT assays were performed to assess cellular viability through monitoring the absorbance of formazan crystals at 570 nm. (C) U2-OS and (D) Saos-2 cells were transfected with control (si-NC) or TRIM21-targeting siRNA in 6-well plates overnight. The cells were treated as described in A and B. The absorbance of the untreated control group was set to 100%. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
-
Next, we identified TRIM21-interacting partners in U2-OS cells using co-IP coupled with LC-MS/MS analysis. To this end, U2-OS cells stably expressing H125-TRIM21 were employed, where the H125-TRIM21 expression system was recombinant lentivirus-based and regulated by a tetracycline (TET) response element (Figure 2A). After induction with TET (10 mg/mL) for 18 h, control U2-OS cells stably expressing the H125-Vector displayed strong green fluorescence (Figure 2B), whereas U2-OS cells stably expressing H125-TRIM21 produced large amounts of TRIM21 protein (Figure 2C).
Figure 2. Introduction of recombinant lentivirus vector expressing TRIM21 (H125-TRIM21) and co-IP assay prepared for LC-MS/MS analysis. (A) Schematic of the construction of recombinant lentivirus vector expressing TRIM21 (H125-TRIM21). (B) Expression of GFP by H125-Vector (H125-V)-transfected cells. U2-OS cells stably expressing H125-V were incubated with tetracycline (TET; 10 mg/mL) for 18 hours. The cells were then visualized using an inverted fluorescence microscope (Olympus, Japan) with a 20 × objective. (C) TRIM21 protein levels in U2-OS cells stably expressing H125-TRIM21. U2-OS cells stably expressing H125-TRIM21 were incubated with TET (10 mg/mL) for 18 hours and then analyzed by Western Blotting with TRIM21 and GAPDH antibodies. (D) Co-IP assay was used to identify the interacting partners of TRIM21. U2-OS cells stably expressing H125-V or H125-TRIM21 incubated with TET (10 mg/mL) for 18 hours were used in standard co-IP assay with control IgG or TRIM21 antibody. The resulting immune complexes containing IgG or TRIM21 antibody were subsequently subjected to in-solution digestion and analyzed by LC-MS/MS.
U2-OS cells stably expressing H125-TRIM21 were used in co-IP with TRIM21 antibody (Figure 2D). Proteins that co-precipitated with TRIM21 were identified by LC-MS/MS. The top three identified proteins are listed in Table 1. The top TRIM21-interacting partner was YWHAZ, which is overexpressed and acts as oncogene to promote proliferation in various tumors[21, 22]. However, there have been few reports on the role of YWHAZ in osteosarcoma. In the present study, to investigate the mechanism of TRIM21 as a positive regulator of U2-OS and Saos-2 cell proliferation, YWHAZ protein was selected for subsequent experiments.
Table 1. Top Three TRIM21 Protein-interacting Partners Identified in this Study
Accession Gene Names Protein Names Molecular Weight (kD) Total Number of Peptides Assigned Sequence Coverage (%) Protein False Discovery Rate Confidence P63104 YWHAZ 14-3-3 protein zeta/delta 27.7 3 12.6531 High P26641 EEF1G Elongation factor 1-gamma 50.1 2 6.4073 High P17066 HSPA6 Heat-shock 70 kD protein 6 71.0 2 6.0653 High -
To confirm the interaction between TRIM21 and YWHAZ, co-IP of U2-OS cells transfected with HA-vector or HA-TRIM21 plasmid were performed. The interacting proteins bound by HA antibody were subsequently analyzed by immunoblot (IB) assay. As shown in Figure 3A, YWHAZ bound to TRIM21.
Figure 3. Interaction between TRIM21 and YWHAZ. (A) Co-IP assay was used to validate the interaction between TRIM21 and YWHAZ. U2-OS cells transfected with HA-vector or HA-TRIM21 plasmid were employed to carry out Co-IP assay with HA antibody. The immune complexes were analyzed by immunoblotting with TRIM21 and YWHAZ antibodies. (B) BIFC assay. U2-OS cells were co-transfected with pHA-VC155 (VC-V), pMyc-VN155 (VN-V), VC-TRIM21, or VN-YWHAZ as indicated in B. After 24 hours, the fluorescence was visualized using an inverted fluorescence microscope with a 20 × objective. (C) Confocal microscopy assay. TRIM21 and YWHAZ were immunostained and then visualized using a laser scanning confocal microscope. DAPI staining was used to assess the morphology of cell nuclei. In the merged panel, yellow represents co-localization of two proteins. Scale bar: 10 μm.
BiFC was then employed to further confirm the interaction between TRIM21 and YWHAZ. BiFC is a method used to observe protein-protein interactions in live cells[33]. BiFC vector plasmids, including pHA-VC155 and pMyc-VN155, were used to construct VC-TRIM21 and VN-YWHAZ, respectively. Interactions between TRIM21 and YWHAZ would promote linking of the complementary non-fluorescent fragments VC and VN, which produces Venus fluorescence. As shown in Figure 3B, 24 hours after transfection of U2-OS cells with VC-TRIM21 and VN-YWHAZ, fluorescence was only detected in cells co-transfected with VC-TRIM21 and VN-YWHAZ plasmids, suggesting binding of YWHAZ to TRIM21. Co-localization of TRIM21 with YWHAZ was then examined using confocal microscopy. As shown in Figure 3C, both TRIM21 and YWHAZ were mainly located in the cytoplasm within scattered granules, where co-localization of TRIM21 with YWHAZ is indicated in yellow in the merged panels. Taken together, these results suggest TRIM21 interacted with YWHAZ and these proteins co-localized in the cytoplasm.
-
TRIM21 is an E3 ligase; therefore, we next investigated whether YWHAZ expression is regulated by TRIM21. Knockdown of TRIM21 with its siRNA resulted in a marked increase in YWHAZ levels (Figure 4A), whereas overexpression of TRIM21 after tetracycline induction resulted in a large decrease in YWHAZ levels (Figure 4B). Accordingly, the addition of proteasome inhibitor MG132 abrogated this decrease (Figure 4B). These results hint at the possibility TRIM21 regulated YWHAZ levels through a proteasome pathway.
Figure 4. RING domain of TRIM21 required for negative regulation of YWHAZ expression by TRIM21. (A) Knockdown of TRIM21 led to upregulation of YWHAZ expression. U2-OS cells were transfected with control (si-NC) or TRIM21-targeting short interfering RNA and then immunoblotted with the indicated antibodies. (B) Downregulation of YWHAZ induced by TRIM21 overexpression was inhibited by the proteasome inhibitor MG132. U2-OS cells stably expressing H125-V or H125-TRIM21 after induction with TET (10 mg/mL) for 18 hours were left untreated or treated with MG132 and then immunoblotted with the indicated antibodies. (C) Diagram displaying full-length TRIM21 and the TRIM21-ΔRING mutant. (D) Effect of the TRIM21 RING domain on YWHAZ expression. U2-OS cells were transfected with control HA-V, HA-TRIM21, or HA-TRIM21-ΔRING and then immunoblotted with the indicated antibodies.
The RING-finger domain of the TRIM protein contains conserved cysteine and histidine residues in a 'cross-brace' arrangement. The RING domain is necessary for recruiting ubiquitin-conjugating enzymes (E2 Ub) and possesses ubiquitin E3 ligase activity[35]. We then determined whether this RING domain takes part in TRIM21 regulation of YWHAZ. To this end, we constructed the HA-TRIM21-ΔRING mutant as shown in Figure 4C. Unlike the plasmid expressing full-length HA-TRIM21, the HA-TRIM21-ΔRING mutant failed to reduce YWHAZ expression (Figure 4D), indicating the RING domain of TRIM21 was required for TRIM21-mediated negative regulation of YWHAZ.
-
Subsequently, we tested whether YWHAZ is involved in TRIM21 regulation of osteosarcoma cell proliferation. Based on our results showing that TRIM21 negatively regulated the expression level of YWHAZ, we investigated whether YWHAZ overexpression could antagonize the effects of TRIM21 on osteosarcoma cell proliferation. As shown in Figure 5A, overexpression of either HA-TRIM21 or Flag-YWHAZ alone, but not the HA-TRIM21-ΔRING mutant, significantly increased U2-OS cell viability. However, overexpression of Flag-YWHAZ failed to antagonize the effects of TRIM21. Moreover, co-expression of HA-TRIM21 and Flag-YWHAZ notably increased cell viability. Intriguingly, similar results were also obtained for cells co-expressing HA-TRIM21-ΔRING and Flag-YWHAZ (Figure 5A, upper panel). The protein levels in these cohorts are shown in the lower panel of Figure 5A. In agreement with the results in Figure 4, overexpression of full-length HA-TRIM21, but not the HA-TRIM21-ΔRING mutant, reduced Flag-YWHAZ levels. However, overexpression of Flag-YWHAZ had no effect on TRIM21 expression (Figure 5B).
Figure 5. Effects of YWHAZ overexpression on TRIM21 overexpression-induced osteosarcoma cell proliferation. (A) U2-OS cells were co-transfected with HA-TRIM21 or HA-TRIM21-ΔRING and Flag-YWHAZ as indicated and evaluated by MTT assays (upper panel) and Western Blotting (lower panel). Results for control cells co-transfected with HA-vector and Flag-vector were set at 100%. The asterisk indicates a statistically significant difference between the indicated cells and control cells. *: P < 0.05; **: P < 0.01; ***: P < 0.001. ###: P < 0.001. (B) U2-OS cells were transfected with Flag-vector or Flag-YWHAZ as indicated and performed standard IB assay with the TRIM21 and Flag antibodies. (C) U2-OS cells were co-transfected with HA-TRIM21 combined with Flag-YWHAZ as indicated and performed standard IB assay with antibodies against HA, Flag, or PCNA.
In addition, PCNA (proliferating cell nuclear antigen), a marker of cell proliferation[36], was used to evaluated the proliferation of U2-OS cells. As shown in Figure 5C, PCNA expression was upregulated in U2-OS cells overexpressing either HA-TRIM21 or Flag-YWHAZ alone compared to the control group. Furthermore, co-expression of both HA-TRIM21 and Flag-YWHAZ caused an obvious re-regulation of PCNA (Figure 5B), which is similar to the trend in cell proliferation observed in Figure 5A. Taken together, our results suggest that YWHAZ was not involved in TRIM21 regulation of osteosarcoma cell proliferation.
doi: 10.3967/bes2018.024
YWHAZ Binds to TRIM21 but Is Not Involved in TRIM21-stimulated Osteosarcoma Cell Proliferation
-
Abstract:
Objective Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21 (TRIM21) in osteosarcoma cell proliferation. Methods 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation (BIFC) were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ. Results TRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However, overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21. Conclusion Our results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma. -
Key words:
- TRIM21 /
- YWHAZ /
- Osteosarcoma /
- Proliferation
-
Figure 1. The effects of TRIM21 on osteosarcoma cell proliferation. (A) U2-OS and (B) Saos-2 cells were transfected with HA-TRIM21 or HA-vector plasmid in 6-well plates and incubated overnight. The cells were then resuspended in McCoy's 5A medium containing fetal bovine serum and seeded into 96-well plates. The cells were then treated with CDDP (30 μg/mL), TRAIL (50 ng/mL), or serum starvation for 24 hours as indicated. MTT assays were performed to assess cellular viability through monitoring the absorbance of formazan crystals at 570 nm. (C) U2-OS and (D) Saos-2 cells were transfected with control (si-NC) or TRIM21-targeting siRNA in 6-well plates overnight. The cells were treated as described in A and B. The absorbance of the untreated control group was set to 100%. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 2. Introduction of recombinant lentivirus vector expressing TRIM21 (H125-TRIM21) and co-IP assay prepared for LC-MS/MS analysis. (A) Schematic of the construction of recombinant lentivirus vector expressing TRIM21 (H125-TRIM21). (B) Expression of GFP by H125-Vector (H125-V)-transfected cells. U2-OS cells stably expressing H125-V were incubated with tetracycline (TET; 10 mg/mL) for 18 hours. The cells were then visualized using an inverted fluorescence microscope (Olympus, Japan) with a 20 × objective. (C) TRIM21 protein levels in U2-OS cells stably expressing H125-TRIM21. U2-OS cells stably expressing H125-TRIM21 were incubated with TET (10 mg/mL) for 18 hours and then analyzed by Western Blotting with TRIM21 and GAPDH antibodies. (D) Co-IP assay was used to identify the interacting partners of TRIM21. U2-OS cells stably expressing H125-V or H125-TRIM21 incubated with TET (10 mg/mL) for 18 hours were used in standard co-IP assay with control IgG or TRIM21 antibody. The resulting immune complexes containing IgG or TRIM21 antibody were subsequently subjected to in-solution digestion and analyzed by LC-MS/MS.
Figure 3. Interaction between TRIM21 and YWHAZ. (A) Co-IP assay was used to validate the interaction between TRIM21 and YWHAZ. U2-OS cells transfected with HA-vector or HA-TRIM21 plasmid were employed to carry out Co-IP assay with HA antibody. The immune complexes were analyzed by immunoblotting with TRIM21 and YWHAZ antibodies. (B) BIFC assay. U2-OS cells were co-transfected with pHA-VC155 (VC-V), pMyc-VN155 (VN-V), VC-TRIM21, or VN-YWHAZ as indicated in B. After 24 hours, the fluorescence was visualized using an inverted fluorescence microscope with a 20 × objective. (C) Confocal microscopy assay. TRIM21 and YWHAZ were immunostained and then visualized using a laser scanning confocal microscope. DAPI staining was used to assess the morphology of cell nuclei. In the merged panel, yellow represents co-localization of two proteins. Scale bar: 10 μm.
Figure 4. RING domain of TRIM21 required for negative regulation of YWHAZ expression by TRIM21. (A) Knockdown of TRIM21 led to upregulation of YWHAZ expression. U2-OS cells were transfected with control (si-NC) or TRIM21-targeting short interfering RNA and then immunoblotted with the indicated antibodies. (B) Downregulation of YWHAZ induced by TRIM21 overexpression was inhibited by the proteasome inhibitor MG132. U2-OS cells stably expressing H125-V or H125-TRIM21 after induction with TET (10 mg/mL) for 18 hours were left untreated or treated with MG132 and then immunoblotted with the indicated antibodies. (C) Diagram displaying full-length TRIM21 and the TRIM21-ΔRING mutant. (D) Effect of the TRIM21 RING domain on YWHAZ expression. U2-OS cells were transfected with control HA-V, HA-TRIM21, or HA-TRIM21-ΔRING and then immunoblotted with the indicated antibodies.
Figure 5. Effects of YWHAZ overexpression on TRIM21 overexpression-induced osteosarcoma cell proliferation. (A) U2-OS cells were co-transfected with HA-TRIM21 or HA-TRIM21-ΔRING and Flag-YWHAZ as indicated and evaluated by MTT assays (upper panel) and Western Blotting (lower panel). Results for control cells co-transfected with HA-vector and Flag-vector were set at 100%. The asterisk indicates a statistically significant difference between the indicated cells and control cells. *: P < 0.05; **: P < 0.01; ***: P < 0.001. ###: P < 0.001. (B) U2-OS cells were transfected with Flag-vector or Flag-YWHAZ as indicated and performed standard IB assay with the TRIM21 and Flag antibodies. (C) U2-OS cells were co-transfected with HA-TRIM21 combined with Flag-YWHAZ as indicated and performed standard IB assay with antibodies against HA, Flag, or PCNA.
Table 1. Top Three TRIM21 Protein-interacting Partners Identified in this Study
Accession Gene Names Protein Names Molecular Weight (kD) Total Number of Peptides Assigned Sequence Coverage (%) Protein False Discovery Rate Confidence P63104 YWHAZ 14-3-3 protein zeta/delta 27.7 3 12.6531 High P26641 EEF1G Elongation factor 1-gamma 50.1 2 6.4073 High P17066 HSPA6 Heat-shock 70 kD protein 6 71.0 2 6.0653 High -
[1] Anderson ME. Update on Survival in Osteosarcoma. Orthop Clin North Am, 2016; 47, 283-92. doi: 10.1016/j.ocl.2015.08.022 [2] Berner K, Hall KS, Monge OR, et al. Prognostic factors and treatment results of high-grade osteosarcoma in norway:a scope beyond the 'classical' patient. Sarcoma, 2015; 2015, 516843. https://www.hindawi.com/journals/sarcoma/2015/516843/fig3/ [3] Ferrari S, Serra M. An update on chemotherapy for osteosarcoma. Expert Opin Pharmacother, 2015; 16, 2727-36. doi: 10.1517/14656566.2015.1102226 [4] Broadhead ML, Clark JC, Myers DE, et al. The molecular pathogenesis of osteosarcoma:a review. Sarcoma, 2011; 2011, 959248. http://www.oalib.com/paper/3080985 [5] Weissman AM. Regulating protein degradation by ubiquitination. Immunol Today, 1997; 18, 189-98. doi: 10.1016/S0167-5699(97)84666-X [6] Hatakeyama S. TRIM proteins and cancer. Nat Rev Cancer, 2011; 11, 792-804. doi: 10.1038/nrc3139 [7] Ozato K, Shin DM, Chang TH, et al. TRIM family proteins and their emerging roles in innate immunity. Nat Rev Immunol, 2008; 8, 849-60. doi: 10.1038/nri2413 [8] Zhang Z, Bao M, Lu N, et al. The E3 ubiquitin ligase TRIM21 negatively regulates the innate immune response to intracellular double-stranded DNA. Nat Immunol, 2013; 14, 172-8. https://www.sigmaaldrich.com/catalog/papers/23222971 [9] Reddy BA, van der Knaap JA, Bot AG, et al. Nucleotide biosynthetic enzyme GMP synthase is a TRIM21-controlled relay of p53 stabilization. Mol Cell, 2014; 53, 458-70. doi: 10.1016/j.molcel.2013.12.017 [10] McEwan WA, James LC. TRIM21-dependent intracellular antibody neutralization of virus infection. Prog Mol Biol Transl Sci, 2015; 129, 167-87. doi: 10.1016/bs.pmbts.2014.10.006 [11] Kyriakidis NC, Kapsogeorgou EK, Gourzi VC, et al. Toll-like receptor 3 stimulation promotes Ro52/TRIM21 synthesis and nuclear redistribution in salivary gland epithelial cells, partially via type Ⅰ interferon pathway. Clin Exp Immunol, 2014; 178, 548-60. doi: 10.1111/cei.2014.178.issue-3 [12] Nguyen JQ, Irby RB. TRIM21 is a novel regulator of Par-4 in colon and pancreatic cancer cells. Cancer Biol Ther, 2017; 18, 16-25. doi: 10.1080/15384047.2016.1252880 [13] Muller J, Maurer V, Reimers K, et al. TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro. Int J Oncol, 2015; 47, 1634-46. doi: 10.3892/ijo.2015.3169 [14] Wada K, Niida M, Tanaka M, et al. Ro52-mediated monoubiquitination of IKK{beta} down-regulates NF-{kappa}B signalling. J Biochem, 2009; 146, 821-32. doi: 10.1093/jb/mvp127 [15] Gao X, Xu F, Zhang HT, et al. PKCalpha-GSK3beta-NF-kappaB signaling pathway and the possible involvement of TRIM21 in TRAIL-induced apoptosis. Biochem Cell Biol, 2016; 94, 256-64. doi: 10.1139/bcb-2016-0009 [16] Jauharoh SN, Saegusa J, Sugimoto T, et al. SS-A/Ro52 promotes apoptosis by regulating Bcl-2 production. Biochem Biophys Res Commun, 2012; 417, 582-7. doi: 10.1016/j.bbrc.2011.12.010 [17] Shibata N, Ohoka N, Sugaki Y, et al. Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders. J Biol Chem, 2015; 290, 28428-37. doi: 10.1074/jbc.M115.670901 [18] Espinosa A, Zhou W, Ek M, et al. The Sjogren's syndrome-associated autoantigen Ro52 is an E3 ligase that regulates proliferation and cell death. J Immunol, 2006; 176, 6277-85. doi: 10.4049/jimmunol.176.10.6277 [19] Ding Q, He D, He K, et al. Downregulation of TRIM21 contributes to hepatocellular carcinoma carcinogenesis and indicates poor prognosis of cancers. Tumour Biol, 2015; 36, 8761-72. doi: 10.1007/s13277-015-3572-2 [20] Brauner S, Zhou W, Backlin C, et al. Reduced expression of TRIM21/Ro52 predicts poor prognosis in diffuse large B-cell lymphoma patients with and without rheumatic disease. J Intern Med, 2015; 278, 323-32. doi: 10.1111/joim.2015.278.issue-3 [21] Xue M, Tao W. Upregulation of MUC1 by its novel activator 14-3-3zeta promotes tumor invasion and indicates poor prognosis in lung adenocarcinoma. Oncol Rep, 2017; 38, 2637-46. doi: 10.3892/or.2017.5948 [22] Obsilova V, Kopecka M, Kosek D, et al. Mechanisms of the 14-3-3 protein function:regulation of protein function through conformational modulation. Physiol Res, 2014; 63, S155-64. http://www.academia.edu/12485797/Mechanisms_of_the_14-3-3_protein_function_Regulation_of_protein_function_through_conformational_modulation [23] Wu YJ, Jan YJ, Ko BS, et al. Involvement of 14-3-3 Proteins in Regulating Tumor Progression of Hepatocellular Carcinoma. Cancers (Basel), 2015; 7, 1022-36. doi: 10.3390/cancers7020822 [24] Watanabe N, Komatsu S, Ichikawa D, et al. Overexpression of YWHAZ as an independent prognostic factor in adenocarcinoma of the esophago-gastric junction. Am J Cancer Res, 2016; 6, 2729-36. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126287 [25] Ruenauver K, Menon R, Svensson MA, et al. Prognostic significance of YWHAZ expression in localized prostate cancer. Prostate Cancer Prostatic Dis, 2014; 17, 310-4. doi: 10.1038/pcan.2014.32 [26] Nishimura Y, Komatsu S, Ichikawa D, et al. Overexpression of YWHAZ relates to tumor cell proliferation and malignant outcome of gastric carcinoma. Br J Cancer, 2013; 108, 1324-31. doi: 10.1038/bjc.2013.65 [27] Neal CL, Yao J, Yang W, et al. 14-3-3zeta overexpression defines high risk for breast cancer recurrence and promotes cancer cell survival. Cancer Res, 2009; 69, 3425-32. doi: 10.1158/0008-5472.CAN-08-2765 [28] Gao X, Feng J, He Y, et al. hnRNPK inhibits GSK3beta Ser9 phosphorylation, thereby stabilizing c-FLIP and contributes to TRAIL resistance in H1299 lung adenocarcinoma cells. Sci Rep, 2016; 6, 22999. doi: 10.1038/srep22999 [29] Gao X, Wang JY, Gao LM, et al. Identification and analysis of glycogen synthase kinase 3 beta1 interactome. Cell Biol Int, 2013; 37, 768-79. doi: 10.1002/cbin.v37.8 [30] Varjosalo M, Sacco R, Stukalov A, et al. Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS. Nat Methods, 2013; 10, 307-14. doi: 10.1038/nmeth.2400 [31] Gao X, Dan S, Xie Y, et al. 14-3-3ζ reduces DNA damage by interacting with and stabilizing proliferating cell nuclear antigen. J Cell Biochem, 2015; 116, 158-69. doi: 10.1002/jcb.v116.1 [32] Huang WS, Xu FM, Zeng QZ, et al. ERK1/2-mediated Cytoplasmic Accumulation of hnRNPK Antagonizes TRAIL-induced Apoptosis through Upregulation of XIAP in H1299 Cells. Biomed Environ Sci, 2017; 30, 473-81. http://www.sciencedirect.com/science/article/pii/S0895398817300818 [33] Pratt EP, Owens JL, Hockerman GH, et al. Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells. Methods Mol Biol, 2016; 1474, 153-70. doi: 10.1007/978-1-4939-6352-2 [34] Kodama Y, Hu CD. Bimolecular fluorescence complementation (BiFC):a 5-year update and future perspectives. Biotechniques, 2012; 53, 285-98. doi: 10.2144/000113943 [35] Joazeiro CA, Weissman AM. RING finger proteins:mediators of ubiquitin ligase activity. Cell, 2000; 102, 549-52. doi: 10.1016/S0092-8674(00)00077-5 [36] Kubben FJ, Peeters-Haesevoets A, Engels LG, et al. Proliferating cell nuclear antigen (PCNA):a new marker to study human colonic cell proliferation. Gut, 1994; 35, 530-5. doi: 10.1136/gut.35.4.530 [37] Lau JM, Wu C, Muslin AJ. Differential role of 14-3-3 family members in Xenopus development. Dev Dyn, 2006; 235, 1761-76. doi: 10.1002/dvdy.v235:7 [38] Muslin AJ, Lau JM. Differential functions of 14-3-3 isoforms in vertebrate development. Curr Top Dev Biol, 2005; 65, 211-28. [39] Joshi S, Yang J, Wang Q, et al. 14-3-3zeta loss impedes oncogene-induced mammary tumorigenesis and metastasis by attenuating oncogenic signaling. Am J Cancer Res, 2017; 7, 1654-64. http://www.ajcr.us/files/ajcr0026751.pdf