Cell culture, transfection and antibodies
The human glioblastoma cell lines U251 and LN229 came from the Institute of Biochemistry and Cell Biology (Shanghai, China). HEK293 cells were from the Institute of Biochemistry and Cell Biology (Shanghai, China). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), and cultured at 37 °C in 5% CO2. We established an IKBKE-shRNA lentiviral vector from GeneChem (Shanghai, China) with the sequence of 5ʹ-GCATCATCGAACGGCTAAATA-3ʹ. A GFP scrambled lentiviral vector with the sequence of 5ʹ-TTCTCCGAACGTGTCACGTTTC-3ʹ was used as the negative control. The shRNAs were transfected according to the manufacturer’s instructions. The Flag-IKBKE plasmid was purchased from Addgene (USA). The HA-TEAD2 and HA-YAP1 plasmids were from Hanbio Biotechnology (Shanghai, China). The IKBKE-overexpress lentivirus and its empty vector virus were from GeneChem (Shanghai, China). We selected Lipofectamine 3000 (thermo fisher scientific, USA) as transfection medium and the process of transfection was according to the manufacturer’s instructions. IKBKE rabbit mAb (No.2905,WB 1:1000; IP 1:100), c-myc rabbit mAb (No.13987,WB 1:1000), MMP9 rabbit mAb (No.13667,WB 1:1000), YAP1 mouse mAb (No.12395,WB 1:1000; IHC 1:400; IP 1:100), Bcl-2 rabbit mAb (No.2870,WB 1:1000), Phospho-YAP (Ser127) rabbit mAb (No.13008,WB 1:1000; IHC 1:2000), HA-Tag rabbit mAb (No.3724,WB 1:1000; IP 1:50), DYKDDDDK-Tag (Flag) rabbit mAb (No.14793,WB 1:1000; IP 1:50) and Axl rabbit mAb (No.8661,WB 1:1000;IHC 1:300) were purchased from Cell Signaling Technology (USA). IKBKE rabbit polyclonal antibody (ab7891,IHC 1:100), Cdk1 rabbit mAb (ab133327,WB 1:10000;IHC 1:300), Cdc25c rabbit mAb (ab32444,WB 1:2000), caspase-9 rabbit mAb (ab202068,WB 1:2000), Bax rabbit mAb (ab32503,WB 1:2000), CyclinB1 rabbit mAb (ab32053,WB 1:5000;IHC 1:100), CyclinA2 rabbit mAb (ab181591,WB 1:2000), c-myc rabbit mAb (ab32072,IHC 1:500), CyclinD1 rabbit mAb (ab134175,WB:1:10000), TEAD2 rabbit polyclonal antibody (ab83670, WB 1:500) were purchased from Abcam (USA). TEAD2 rabbit polyclonal antibody (sc-67115, IP 1:50) was from Santa Cruz (USA). LATS2 rabbit polyclonal antibody (20276-1-AP, WB 1:500; IHC 1:50) were purchased from proteintech (USA). GAPDH mouse mAb (WB 1:2000) was from ZSGB-Bio (Beijing, China).
Protein extraction and western blot analysis
The cell total protein was extracted after treatment of CYT387, plasmids or IKBKE-shRNA using RIPA lysis buffer with protease and phosphatase inhibitor (MCE USA). The homogenates were clarified by centrifugation at 4 °C for 15 min at 12,000 rpm after cleavage by RIPA for 15 min, and the protein concentration was measured by BCA assay kit (Beyotime, Shanghai, China). At least 20 μg protein mixed with 4 × loading buffer was added into spacer gel and then separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein bands were electrotransferred to PVDF membranes (Millipore, USA). Primary antibodies were incubated at 4 °C for overnight then HRP-conjugated secondary antibody (1:3000 dilution, ZSGB-Bio, Beijing, China) was used for 1 h at room temperature. The bands were detected by the G:BOX (Syngene Company, UK) using Chemiluminescent HRP Substrate (Millipore USA).
RNA extraction and real-time RT-PCR analysis
Total RNA of glioblastoma cells (U87-MG and LN-229) after treatment with CYT387 in dose- and time-dependent manners was extracted by TRIzol reagent (Invitrogen, USA) following the manufacturer’s protocols and then reverse transcription was performed using GoScript™ Reverse Transcription System (Promega, USA). The quantitative real-time PCR was finished by GoTaq qPCR Master Mix (Promega, USA) according to the supplier’s instructions. The reaction conditions were as follows: 95 °C for 5 min and 40 cycles of 95 °C for 12 s and 60 °C for 40 s. The primers were synthesized by GENEWIZ (USA). The sequences of the primers were as follows: GAPDH: 5ʹ-GGAGCGAGATCCCTCCAAAAT-3ʹ (Forward primer) and 5ʹ-GGCTGTTGTCATACTTCTCATGG-3ʹ (Reverse primer); IKBKE: 5ʹ-GAGAAGTTCGTCTCGGTCTATGG-3ʹ (Forward primer) and 5ʹ-TGCATGGTACAAGGTCACTCC-3ʹ (Reverse primer); TEAD2: 5ʹ-GCCTCCGAGAGCTATATGATCG-3ʹ (Forward primer) and 5ʹ-TCACTCCGTAGAAGCCACCA-3ʹ (Reverse primer); YAP1: 5ʹ-TAGCCCTGCGTAGCCAGTTA-3ʹ (Forward primer) and 5ʹ-TCATGCTTAGTCCACTGTCTGT-3ʹ (Reverse primer). GAPDH was used as internal control.
Clone formation assay
U87-MG and LN-229 cells were seed in six-well plates (2 × 103/well) divided into three groups as blank control, negative control (DMSO) and drug group (CYT387 with concentration of 6 μM). Growth medium was changed every 6 days. After 12 days, cells were fixed in 4% paraformaldehyde for 15 min and stained with crystal violet for 30 min. Colonies were scored after photographed.
For IC50 measurement, we seeded U87-MG and LN-229 cells (5000/50 ul/well) into 96-well plates on the first day. After cells were adherent, we again added 50ul/well medium with different concentration of CYT387 to achieve the final drug concentration gradient of 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM and 64 μM. After treatment with CYT387 for 24, 48, and 72 h, 10 μl CCK8 reagent (dojindo, Japan) was mixed into each well and then 96-well plate was incubated for 2 h at 37˚C. The O.D. value was measured by Microplate reader at the wavelength of 450 nm.
For proliferative curve measurement, glioblastoma cells with normal medium, DMSO medium and 6 μM CYT387 medium were seeded (2000/100 µl/well) into 96-well plates. From first day to fifth day, 10 µl CCK-8 reagent (dojindo, Japan) was added into each well. The O.D. value was measured after incubated for 2 h at 37 °C in a 5% CO2 atmosphere.
Wound healing assays
U87-MG and LN-229 cells were seeded in six-well plate and a straight wound was created with a sterile 100 µl pipette tip. Then we respectively added DMSO and CYT387 in DMSO group and drug group, making the drug concentration of 6 μM. After treatment for 24 h and 48 h, the wound healing area was detected by an inverted microscope.
Before experiment begun, matrigel with 3 times volume serum-free DMEM (total 80 µl/well) was coated on the upper surface of chamber. Then place it at 37 °C for 30 min, waiting for matrigel solidification. Then the cells (5 × 104/well) were seeded into the transwell chambers with 200 μl serum-free DMEM while outer space was filled with 500 μl serum DMEM. After incubated at 37 °C in a 5% CO2 atmosphere for 48 h, cells across the chamber membrane was fixed with 4% paraformaldehyde for 15 min, then stained with crystal violet for 5 min, counted and imaged under the microscope.
Cell apoptosis assays and cell cycle analysis
Before cell apoptosis assay, the cells were treated with CYT387 with concentration of 6 μM for 24 h. Cells were trypsinized without EDTA, washed with PBS twice and then stained using the Annexin V-FITC Apoptosis Detection kit from KeyGen Biotech (Nanjing, China) according to the manufacturer’s instructions. Flow cytometry analysis was finished by a FACS flow cytometer (Becton–Dickinson). Data were analyzed by CellQuest software. Before cell cycle analysis, the U87-MG and LN-229 cells was treated with CYT387 at the concentration of 6 μM for 72 h. Then the following protocols were performed as the previous article .
Co- immunoprecipitation (co-IP) was carried out as described previously .
Before animal studies, CYT387 purchased from selleck (USA) was dissolved in NMP (1-methyl-2-pyrrolidinone) to finally get the concentration of 120 mg/ml. Next, the CYT387 was diluted with 0.14 M Captisol (MCE, USA) to a concentration of 6 mg/ml. The tumor subcutaneous experiments method was carried out as described previously . After subcutaneous tumour was shaped, the nude mice were fed with CYT387 (100 mg/kg/day).
For intracranial orthotopic model, U87-MG transfected with luciferase-expressing lentivirus was injected intracranially into 6-week-old BALB/c-nu mice. After 7 days, mice started to be fed with CYT387 (100 mg/kg/day). The weight of mice was monitored every 2 days and the luminescence imaging of intracranial tumour was measured using an IVIS Lumina Imaging System (Xenogen) every 7 days.
After mice were sacrificed for subcutaneous and intracranial tumour, we made specimens embedded with paraffin. Then the paraffin-embedded tumours were sectioned and dewaxed. After antigen retrieval using 10 mmol/l citrate buffer, sections were incubated with 3% H2O2 and blocked with 5% BSA. Then the sections were added with primary antibodies at 4 °C overnight. After rewarming at room temperature the next day, the sections were incubated with secondary antibodies using two-step polymer HRP detection system (ZSGB-BIO, Beijing, China). The samples were colourated with DBA Kit (ZSGB-BIO, Beijing, China) and then counterstained with haematoxylin. After dehydration and sealing piece with neutral gum, the samples were detected and photographed by microscope (Olympus Japan).
All data were repeated at least three times. Quantitative data are shown as the mean ± standard deviation (SD). We used SPSS software (version 16.0) for the statistical analyses and P < 0.05 was considered statistically significant.