2.1 Cell culture
U87, U251 of the cell lines for human GBM were gotten in the cell bank type culture collection of the chinese academy of sciences. identified technology of our cell lines in the laboratory was repeat profiling of short tandem, provided by Porcello Life Science &Technology Co Ltd. Dulbecco's modified Eagle's medium (DMEM), including 100µg/ml streptomycin, 10% fetal bovine serum (FBS) as well as 100µg/ml penicillin, was applied to incubate the cells at 37℃ under 5% CO2 atmosphere.
2.2 Chemicals and antibodies
UM-164 was purchased from Bio Vision and dimethyl sulfoxide was applied to dissolved it, which was purchased from Merck KGaA. and the antibodies were all from Cell Signaling Technology, Inc., except BCL-2 (GTX100064), which was from Gene Tex, Inc. and cleaved caspase-3 (ab32042), which was purchased from Abcam, Inc. including these, Anti-AKT (#4691), phosphorylated-AKT (#4060), EGFR (#1416), P-EGFR (#3777), P38 (#41666), phosphorylated- P38 (#4511), P42 (#9108), P-P42 (#4370), BCL-XL (#2764), Bax (#5023), cyclin D1 (#2978), cyclin E (#20808), MMP-9 (#13667), MMP-2 (#40994), and GAPDH (#5174).
2.3 Cell viability
The Cell Counting Kit-8 (CCK-8), purchased from dojindo molecular technologies, Inc. was employed to assess the viability of cell and confirm the inhibitory effect of UM-164 to U87 and U251. in a 96-well plate, every well includes 5000 cells, which were planted into a solution included 100µl DMEM containing 10% FBS, as well as UM-164, which was divided in multiple concentrations (0, 1, 5, 10, 50, and 100µM) for 2 days and given a fixed concentration with different times (24, 48, 72 and 96h). then, 10µl CCK-8 was applied to incubate cells for 60 minutes at 37℃. Every group was gauged the absorbance value in each well with a spectrophotometer at 450 nm for 3-time.
2.4 Colony formation assay
Adding U87 and U251 (~ 1,000) cells into 2ml DMEM with 10% FBS and put in a 6-well plate. next, various concentrations of UM-164 (0, 1, 5 and 10µM) were used to treat the cells for 14-day at 37℃ under CO2 of 5%. in 25℃, paraformaldehyde, 2ml 5%, was added in the cells for a quarter. the cells in every well were then stained by 0.5% crystal violet and taken photos by a camera.
2.5 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay
In a 96-well plate, every well includes 5000 cells, planted into a solution included 100µl DMEM, containing 10% FBS, and UM-164, which was divided in some concentrations (0, 1, 5, 10, and 20µM) about 3 days. then, cultivating the cells in 50µM Edu under 5% CO2 at 37℃, as well as fixing them by 4% paraformaldehyde about half an hour at 25℃. we treated the cells in triton X-100, 0.5%, for 20- minute as well as used PBS to wash them 3 times in 5 minutes. the cells were added 100µl 1X Apollo® reaction cocktail to incubate them for 30 minutes. hoechst 33342, 5µg/ml, was employed to stain the cell nuclei 30-minute. getting fluorescence
pictures by using fluorescence microscope in ×200 magnification.
2.6 Cell cycle distribution analysis
Distributions of cell cycle were gotten through flow cytometry, which employed a cell cycle kit of PI staining (Becton, Dickinson and Company). seeding U87 and U251 into 6-well plate and treating them with multiple concentrations (0, 1, 5, 10and 20µM) for 72h. afterward, a centrifuge, at 1000rpm, was employed in 25℃ for 5 minutes to collect cells. PBS and cold 70% ethanol was mixed into the cells to wash them for 1day at 4℃. treating the cells at 37℃ by 50 µl 100 µg/ml RNase, washing two times with PBS, centrifuging about 5 minutes at 1,000 rpm and staining in 5µl PI with stock solution, 50mg/ml. evaluating the outcomes was done by BD FACSAria. And getting assessments of the statistics from ModFit LT 4.0.
2.7 Wound-healing assay
Inoculating U87 as well as U251, 70%~80% concentration, in a plate to become a monolayer. after that, get straight cut lines in every well, we used a pipette tip to scrape respectively. Next, removing the suspended cells with PBS, adding UM-164 with several concentrations (0, 5 or 10µM) and 2ml DMEM. acquiring pictures with a microscope (Olympus BX51; Olympus Corporation) at ×10 magnification, and assessing the wound distance with imageJ software.
2.8 Invasion assays, ant stain with Phalloidin as well as Cell Migration
Transwell chambers were applied with a polycarbonate film with an 8.0µm pore diameter. mixing U87 as well as U251 with DMEM lack of 10% FBS and planting them in the top chambers. then, we used Matrigel to coated the cells, or not, in migration and invasion assay. Later, putting the chambers in a 24-well plate to incubate about 24 hours. After that, we stained the cells with the crystal violet that crossed the membranes, and carefully wiped off the cells having not penetrated membranes. quantifying the cells by using a microscope (magnification, x10; Olympus BX51; Olympus Corporation). planting the cells into 6-well plates, having glass slice at their bottom, and handling them with UM-164 in IC50 concentration. subsequently, incubating the cells with Phalloidin, which was diluted 100 times by BSA, for 1 hour and staining them for 5 minutes by DAPI. then, rinsing the cells twice for 5 minutes. finally, visualizing fluorescence images with a fluorescence microscope (Olympus BX51; Olympus Corporation), ×200 magnification.
2.9 Analyzing apoptosis with flow cytometry
Apoptosis Annexin V-PE/7-AAD kit was used to inspected to cells dealt with different UM-164 concentration (0, 1, 5, 10 and 20µM). firstly, we used centrifugation for 5 minutes (1,000rpm at 25℃) to collect cells and then rinsed them twice with PBS. After that, 1X binding buffer (0.1 mM HEPES/NaOH, 1.4 M NaCl and 25 mM CaCl2, pH 7.4)100µl was used to let the cells float in it. and add PE-Annexin V and 7-ADD both with 5µl, keep in the darkness at 25℃ for 15 minutes to stain the cells. subsequently, sucking 400µl 1X binding buffer into every tube. BD FACSAria (BD Biosciences) was used to analyze the achievements. FlowJo software (FlowJo LLC) was adopted to quantify statistics, and the total apoptosis rates were calculated and evaluated by the totality of the upper right and low right quadrants. in line with manufacturer’s protocol (Roche Molecular Diagnostics), TUNEL assay could be used to get the detection of apoptotic cells’ DNA fragments. Olympus BX51 microscope (Olympus Corporation) was used to get photos.
2.10 Measuring potential (ΔΨm) of mitochondrial membrane
One of the iconic events of early apoptosis is the disappearance of mitochondrial membrane potential (ΔΨm). firstly, planting the cells into 6-well plates, having glass slice at their bottom, and handling them with UM-164. accordance to the instructions of manufacturer. the indication of ΔΨm’s disappearance emerged as a decline of the ratio of red/green fluorescence intensity, which can be found out by flow cytometry and microscopy.
2.11 ROS level of cells treated with UM-164
The ROS level was tested by DCFH-DA. first, planting the cells into 6-well plates with glass slice at their bottom and handling them with UM-164. Then, the cells were conducted with the ROS stain, in line with the explanations of manufacturer. the flow cytometry was employed to detect the raising overall ROS level in cell. capturing images under the usage of Olympus FV1200 confocal microscope or Olympus BX51 microscope.
2.12 Western blot analysis
First, UM-164 (0, 5, 10 or 20µM) was adopted to treat the cells for 48 hours. next, lysing the cells in RIPA buffer on ice for 20 minutes. then, loading the protein onto SDS-PAGE and electro-transferring them into PVDF membranes about sixty or ninety minutes. after that, 5% skim milk was employed to block the membranes at 4°C, membrane and primary antibody were incubated at 1:1000 after one night. alexa fluor 680/790-labelled secondary antibodies, at 1:1000, were put in the membranes to incubate for one hour. the LICOR odyssey infrared imaging system can let the bands visible. and normalizing the data with the usage of GAPDH.
First, fixing the cells in 4% paraformaldehyde for half an hour. Then, permeabilizing cells with Triton X-100 0.1% about 10-minute. Next, 1% bovine serum albumin was applied to block them about one hour. indicated primary antibodies were mixed in them to incubate overnight then adopting fluor-labelled secondary antibodies, purchased from Antgene, Wuhan, China. DAPI was used to stained the nuclei. Olympus FV1200 confocal microscope microscope was applied to get images.
2.14 Statistical analysis
SPSS 19.0 as well as GraphPad Prism 6.0 are used to analyze statistics. indicating all data as mean ± SD. one-way analysis of variance and t-test were used to evaluate the discrepancy between groups. every experiment repeated in triplicate. considering P < 0.05 as having statistically significant.