Supplementary MaterialsAdditional file 1: Table S1

Supplementary MaterialsAdditional file 1: Table S1. B. TKT wild type overexpression promoted the transition from G0/G1 to S phase, and the NLS mutant decrease the cell cycle regulating function of TKT. C. Although TKT enzyme-inactivating mutant D155A reduced the function of regulating cell cycle, the expression of TKT-D155A still increased the percentage of S phase, suggesting there would be a non-metabolic mechanism of TKT to regulate cell cycle. D. TKT-K6R-D155A double mutation would abolish the function of regulating cell cycle. E. The glucose consumption of TKT, NLS mutation and enzyme-inactivating mutation overexpressing cell lines. (PDF 358 kb) 13046_2019_1131_MOESM5_ESM.pdf (359K) GUID:?B3E4CE1C-518D-479F-B559-868060397459 Additional file 6: Figure S4. Workflow and quantity control of the cross-linking Co-IP/MS. A. Cross-linking Co-IP/MS workflow. B. Equally overexpressed TKT wild type and TKT NLS mutation (K6R) stable cell lines along with the empty vector control group were crosslinked by formaldehyde. C. Nucleus fractions were enriched after Briciclib weak power sonication. The marker of nucleus (LAMN B) could only detected in nucleus fraction. D. Wide type TKT, but not TKT NLS mutant could be detected in nucleus fractions. Briciclib E. TKT antibody was used to pull down the target protein after crosslinking. F. Overlap of proteins identified in the 3 stable cell lines by MS. (PDF 177 kb) 13046_2019_1131_MOESM6_ESM.pdf (177K) GUID:?B9A9D8E2-876E-4FFB-A2B6-C46C3FF8F120 Additional file 7: Table S3. The list of 243 unique proteins interacting with nuclear TKT. (DOCX 41 kb) 13046_2019_1131_MOESM7_ESM.docx (41K) GUID:?220513FE-65D4-4C37-812C-2B2629B248AB Data Availability StatementAll Mass Spectrum raw data and the MaxQuant output tables have been deposited to iProX and can be accessed with the iProX accession: IPX0001386000. Abstract Background Metabolic reprogramming is one of the hallmarks of cancer cells. The pentose phosphate pathway (PPP), a branch of glycolysis, is an important metabolic pathway for the survival and biosynthesis of cancer cells. Transketolase (TKT) is usually a key enzyme in the non-oxidative phase of PPP. The mechanistic details of TKT in hepatocellular carcinoma (HCC) development remain unclear. Methods TKT level and subcellular location were examined in HCC cell lines and tissue samples. We established the TKT overexpression and knocking-down stable cells in HCC cell lines. Proliferation, migration, viability and enzyme activity assays in vitro, tumor metastasis and development assays in vivo were employed to check the consequences of TKT on HCC advancement. GFP-tagged TKT truncations and mutants had been used to find the nuclear localization series (NLSs) of TKT. Cross-linking co-IP/MS was put on identify the relationship protein of nuclear TKT. Outcomes We demonstrated that TKT elevated the migration and proliferation of HCC cells, as well as the viability under oxidative stress in vitro and accelerated the growth and metastasis of HCC cells in vivo. We found as a key enzyme of PPP, TKT could promote the proliferation, cell cycle, SLC12A2 migration Briciclib and viability by regulating the metabolic flux. Moreover, it was firstly reported that unlike other key enzymes in PPP, TKT showed a strong nuclear localization in HCC cells. We found not only high TKT expression, but also its nuclear localization was a prediction for poor prognosis of HCC patients. We further identified the nuclear localization sequences (NLS) for TKT and exhibited the NLS mutations decreased the pro-tumor function of TKT independent of the enzyme activity. Cross-linking Co-IP/MS showed that nuclear TKT interacted with kinases and transcriptional coregulators such as EGFR and MAPK3, which are associated with cell activation or stress response processes. EGF treatment significantly increased the viability and proliferation of HCC cells in the enzyme-inactivating mutation TKT-D155A overexpression cells but not in the NLS-D155A double mutant group, which could be blocked by EGFR inhibitor erlotinib treatment. Conclusions Our research suggests that in addition to the metabolic manner, TKT can promote the development of HCC in a non-metabolic manner via its nuclear localization and EGFR pathway. Electronic supplementary material The online version of this article (10.1186/s13046-019-1131-1) contains supplementary material, which is available to authorized users. and [22]. TKT was the majority of transketolase not only in human normal organs but also in most tumor tissues while TKTL1 and TKTL2 are mainly expressed in testis [23, 24]. In the present.