Antibody against CTR9 was purchased from Cell Signaling Technology (Danvers, MA, USA). JAK2, p-JAK2, STAT3, p-STAT3 and β‐actin antibodies were bought from Proteintech (Wuhan, Hubei, China).
Twenty-two fresh brain tissue specimens from glioma and six fresh brain tissue specimens from traumatic brain injury were selected randomly from the specimen repository in Nanjing Brain Hospital. All tissues were taken from the central site of the resected tumors with typical morphology and pathologically confirmed diagnosis.
The selected samples included non-tumor group (6 cases), grade II group (8 cases), grade III group (8 cases) and grade IV group (6 cases), according to the WHO tumor classification standard.
The glioma cell lines U251, T98G and human embryonic kidney cell line HEK293T were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. All cells were cultured in DMEM with10% fetal bovine serum in a 37°C constant temperature incubator containing 5% CO2.
Construction of lentivirus
Three short hairpin RNAs (shRNAs) were designed to knock down CTR9 and synthesized as followings:
Establishment of U251 and T98G cell lines
Control, shCTR9#3, GFP and GFP-CTR9 plasmids were co-transfected with helper plasmids pMD2.G and pSPAX2 in HEK293T cells, respectively. Virus supernatants were collected 48 hours after transfection. U251 and T98G cells were infected by obtained lentiviruses and stably expressed Control, shCTR9#3, GFP and GFP-CTR9 after puromycin filtration.
Cells at the logarithmic phase were inoculated into 96-well plates. EdU assay was conducted after overnight culture. Diluted reagent 5‐ethynyl‐20‐deoxyuridine was added into each well and cells were incubated at 37℃ for 2 hours. The cells were washed with PBS twice and fixed with 4% paraformaldehyde at room temperature for 30 minutes. Afterwards, the cells were incubated with glycine and 0.5% TritonX-100 for 10 and 30 minutes, respectively. Apollo® reagent was added into each well and cells were incubated in dark for 30 minutes. After dealt with 0.5% TritonX-100 and methanol, the nuclei of the cells were dyed with Hoechst for 20 minutes and photographed by a fluorescent microscopy.
Cell viability assay
Cell suspension was inoculated into 96-well plates. CCK-8 assay was conducted after overnight culture. 10 μL CCK-8 reagent was applied to each well (Be careful not to form bubbles in the well) and the absorbance at 450nm was measured with a SynergyMx Multi- Mode Microplate Reader (Biotek, Winooski, VT) after reaction for 4 hours at 37℃.
Transwell migration and invasion assays
To assess invasion ability, the upper chamber of each filter was covered with 10ug Matrigel(BD, San Jose, CA, USA), which was evenly spread and placed in a 37°C incubator for 2 h. The serum-free cell suspension was then added into the fliter and the lower chamber was filled with 10% FBS. After 30 hours of incubation at 37°C, the non-invasive cells were swabbed from the upper chamber. Then cells on the lower side of the filter were fixed with 4% paraformaldehyde for 30 minutes and dyed with 0.03% crystal violet solution for 10 minutes. Three fields of adherent cells in each well were randomly photographed and counted. The same experiment was conducted to assess migration ability except for filters pre-coated with Matrigel.
The same amount of proteins were placed on 10% SDS-PAGE and then transferred to PVDF membrane after the required strips appeared. The membrane was sealed with 3% BSA for 1 hour at room temperature and cut according to the molecular weight. The membranes were placed into tubes containing primary antibodies at 4°C overnight and secondary antibodies at room temperature for 1 hour. The antibodies were detected by ECL or DAB. Image J analysis software was used to analyze the strips.