2.1 Cell lines culture and regents
The human glioma cancer cells line LN229 and U87 were purchased from American Type Culture Collection (ATCC), and cultured with RMPI-1640 complete medium (Gibco, MA, USA), supplemented with 10% feral bovine serum (FBS) (Gibco, MA, USA) at 37°C with 5% CO2. ATN-161 (Ac-PHSCN-NH2) was purchased from Selleck Chemicals (MA, USA). JAK1-IN-3 and STAT3-IN-1 were obtained from MCM (MA, USA). TMZ was purchased from Sangon (Shanghai, China).
2.2 Patients’ tumor tissues samples
Formalin-fixed, paraffin-embedded human glioma tumor tissue sections, lacking any patient-identifying information, were obtained from the Affiliated Hospital of Southwest Medical University, and divided into high degree group (HD, WHO level 3 ~ 4) and low degree group (LD, WHO level 1 ~ 2). The protocols were approved by Regional Scientific Ethics Committee of the Affiliated Hospital of Southwest Medical University. Written informed consent was attained from all subjects, and all methods were performed in accordance with the Declaration of Helsinki.
2.3 Cell proliferation and colony formation assay
The cell proliferation was determined by Cell Counting Kit-8 (CCK-8, Solarbio, Beijing, China). Briefly, 2000 human glioma cancer cells were seeded into 96 well plate and cultured for 72 h at 37°C. Then, 10 µL CCK8 solution was added into each well and incubated for 90 min at 37°C. The cells proliferation was analyzed through the absorbance at 450 nm, measured by a microplate reader (Bio-Rad, Hercules, USA). For cell colony formation assay, LN229 or U87 cells (~ 500) were seeded in a 6-well plate. After 14 days, colonies were fixed with methanol for 10 min and stained with 1% crystal violet (Sigma, MA, USA) for 1 min. Each group was measured in triplicate.
2.3 Preparation of different stiffness PA gel
Different stiffness PA gel used in cell culture were prepared as indicated in previous studies(13). Briefly, we coated the coverslips with a thin layer of gel containing a mixture of 3–10% acrylamide and 0.01%-0.3% bis‐acrylamide, which produced gels of 2, 8 and 20 kPa stiffness. PA gel polymerization was promoted by the addition of 10% APS (1/100 volume) and TEMED (3/1000 volume). Then the coverslips were washed with PBS twice for 20 minutes, followed by sterilizing in PBS solution for 1 hour with ultraviolet light. Next, we added 50 µL heterobifunctional sulphosuccinimidyl 6‐(4′‐azido‐2′‐nitrophe‐nylamino) hexanoate and photo-activated for 5 minutes with ultraviolet light. Finally, the coverslips were coated with 10 µL/mL fibronectin for 1.5 hours and rinsed before cell seeding. Gels were soaked in serum-free culture media for 24 hours for usage. All regents of PA gel were purchased from Solarbio (Beijing, China).
2.4 Atomic force microscopy (AFM) analysis
Atomic force microscopy and analysis were performed as previously described(16).Frozen tissue blocks were cut into 20 µm thick sections. Each section was immersed in PBS and thawed at room temperature before AFM measurement. The samples were maintained in proteinase inhibitor in PBS (protease inhibitor cocktail, Roche 14 Diagnostics, 11836170001) supplemented with Propidium Iodide (SIGMA P4170, 20 µg/ml) during the AFM session. AFM indentations were performed using an MFP3D-BIO inverted optical AFM (Asylum Research) mounted on a Nikon TE2000-U inverted fluorescent microscope, as previously described(17).
2.5 Western blotting
The protein lysates were separated by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membrane was incubated with the primary antibodies against to JAK1, JAK2, STAT3, p-STAT3, p-JAK1, p-JAK2, c-Myc, integrin α5, integrin β1(1:500, Abcam, Cambridge, UK), followed by incubation with an HRP-conjugated secondary antibody (1:1000, Abcam, Cambridge, UK). Beta-actin (1:1000, Abcam, Cambridge, UK) was set as a control.
2.6 Flow cytometry
The LN229 and U87 cells were fixed with 4% PFA (Affymetrix, 19943), and stained with anti-human CD133 (Biolegend, MA, USA) at 4°C for 30 mins, according to the manufacture instructions. All the dates were acquired on an AccuriC6 (BD, MA, USA) and analyzed with FlowJo software.
2.7 Immunofluorescence Staining
The pathological sections of glioma cancer patients’ tumor tissues were retrieved by microwave antigen retrieval (Citrate-EDTA Antigen Retrieval Solution, Beyotime, Beijing, China). After blocked by 5% BSA, the sections were incubated primary antibodies: anti-p-JAK1, anti-p-STAT3, anti-c-Myc (1:200, Abcam, Cambridge, UK) overnight at 4 ºC and followed by signal amplification using the ABC HRP Kit (Thermo, MA, USA), for 2 hours at room temperature, and the nucleus was stained with DAPI. All immunofluorescence images were captured from FV1000 laser scanning confocal microscope (Leica, Barnack, Germany).
2.8 siRNA silence
For c-Myc silenced tumor cells, LN229 and U87 cells were infected with Lipofectamine 8000 (Beyotime, Beijing, China) according to the manufacturer’s protocol. The relevant c-Myc siRNA sequence as followed: siRNA#1: 5'-CTATGACCTCGACTACGACTTCAAGACCGTCCTAGTCGAGGTCATAG-3' and siRNA#2:5'-CCACAGCATACATCCTGTTTCAAGACGACAGGATGTATGCTGTGGC-3'
2.9 Animal experiments
All the animal care and experimental procedures were approved by the Animal Care and Use Committee of the Affiliated Hospital of Southwest Medical University Committee Guidelines on the Use of Live Animals in research, which is according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. LN229 and U87 glioma subcutaneous models were established on female NSG mice at 4 weeks of age, which purchased from Huafukang company (Beijing, China) and raised in SPF level. For tumor analysis, 1x106 LN229 and U87 cells in 100 µL PBS were subcutaneously injected into the right side of NSG mice. All mice were randomly assigned to 2–4 groups (n = 6 per group), and the tumor volume was measured every day for 4 weeks. For tumorigenesis analysis, LN229 or U87 cells (1×105) were subcutaneously injected into the right side of mice. After 2 weeks, tumor numbers on each mouse was measured.
For drugs treatment, 14 days later, mice were treated with 2.5 mg/kg Temozolomide
(TMZ, Sangon, Shanghai, China),10 mg/kg STAT3-IN-1 by oral administration for two weeks (twice a week) respectively. Tumor volume was measured using the formula: volume = length x width2/2. The survival time of mice was recorded.
2.10 Statistical analysis
GraphPad Software (La Jolla, CA) and SPSS 16.0 (SPSS, Chicago, IL, USA) was used for ANOVA, or Student’s t-test. Each experiment was performed in triplicated. Data are expressed as the mean ± standard deviation (SD). Survival time was analyzed by a Log-Rank. P < 0.05 was defined as a statistically significant difference. *P < 0.05, **P < 0.01 and ***P < 0.001, n.s no significant difference.