However, Zhang and his colleagues indicated that p-ERK1/2 is usually a key upstream key factor of p-GSK3. HJURP expression facilitated the proliferation of HCC cells, whereas the depletion of HJURP resulted in decreased cell growth in vitro and in vivo. Furthermore, the effects of HJURP silencing were reversed by p21 knockdown. Likewise, p21 overexpression inhibited cell growth ability mediated by HJURP elevation. Mechanistically, HJURP destabilized p21 via the MAPK/ERK1/2 and AKT/GSK3 pathways, which regulated the nucleus-cytoplasm translocation and ubiquitin-mediated degradation of p21. Clinically, high HJURP expression was correlated with unfavorable prognoses in HCC individuals. Conclusions Our data revealed that HJURP is an oncogene that drives cell cycle progression upstream of p21 in HCC. These findings may provide a potential therapeutic and prognostic target for HCC. forward 5-AGTGCCTTTATGTATTGGAG-3, and reverse 5- AAGTGAGGGTCTGGATTTA-3; forward 5-GCAGACCAGCATGACAGATT-3, and reverse 5-TAAGGCAGAAGATGTAGAGCG-3; and forward 5- GAACATCATCCCTGCCTCTACT-3, and reverse 5- ATTTGGCAGGTTTTTCTAGACG-3. was used as an internal control. Western-blot The total proteins were extracted for 60?min on ice in RIPA buffer (Thermo Scientific, USA) containing protease and phosphatase inhibitors (Cell Signaling Technology, USA). Cell lysates were centrifuged at 1.2??104?g, 4?C for 15?min, and the concentrations of supernatants were detected with a BCA Protein assay kit (Thermo Scientific, USA). 30?g protein was separated by 10% SDS-PAGE (Life Technology, USA) and then transferred to 0.45?m PVDF membranes (Millipore, USA). The membranes were incubated with monoclonal antibodies at 4?C for 24?h. In total, primary antibodies included those for HJURP, ERK1/2, p-ERK1/2, cyclinD1, cyclinE, p-JNK, GSK3, p-GSK3 AKT, p-AKT (Abcam, UK), LRR1 (Proteintech, China) and p21 (Cell Signaling Fluorescein Biotin Technology, USA). The immunoblots were detected with a visual imaging system (Bio-Rad, USA). -actin and GAPDH (Solarbio Life Science, China) were selected as the loading controls. Cell viability assay The cell viability assays were performed with a Cell Counting Kit-8 Assay (DOJINDO Laboratories, Japan). The HCC cells were seeded into 96-well plates (1??103cells per well for the HCC-LM3 and SMMC-7721 cells, and 2??103cells per well for the Huh7 cells) in 100?l medium incubated at 37?C, Fluorescein Biotin 5%CO2 in humidified incubator. After the indicated number of days, the supernatants were removed, 90?l medium and 10?l CCK-8 were added to each well, and the plates were then incubated for 1?h. The absorbance at 450?nm was detected with a microplate reader (BioTek, USA). Cell cycle analysis The HCC cells were collected and fixed using 75% pre-cooled ethanol at 4?C overnight. After being washed three times with phosphate buffered saline (PBS) and resuspended with 300?l DNA staining solution (Multiscience, China) at room heat for 30?min. The cell cycle analysis was detected via a flow cytometry (FACS LSRII, BD Bioscience, USA). Colony formation assay For colony formation assessment, 2??103 stably infected cells were seeded into 6-well plates. After incubation for 15?days, the plates were washed with PBS for three times and 4% paraformaldehyde used to fix the cells for 25?min. Subsequently, the cells were stained with 0.5% crystal violet solution for further counting and statistical analysis. Immunofluorescence assay For immunofluorescence assay, 5??104 stably transfected tumor cells were seeded in a 2?mm confocal Fluorescein Biotin plate (Nunc, USA) for culture in the indicated incubator. Cultured cells were fixed with 4% paraformaldehyde and then permeabilized with 0.25% TritonX-100. After Rabbit Polyclonal to LMO3 blocking with 1% bovine serum albumin (BSA), the primary antibodies against p21 (1:100) (Abcam, USA) were added into each plate for an overnight incubation at 4?C. The secondary antibodies (1:200, Sigma-Aldrich, USA) were incubated with the cells at 37?C for 30?min and DAPI was used to stain the nuclei at 37?C for 10?min. The images were captured by a fluorescence microscope (Olympus BX53, Japan). Nuclear and cytoplasmic protein extraction.