Patients and species
Tissue samples and adjacent normal tissues were obtained from patients who underwent surgery at the First Affiliated Hospital of Guangxi Medical University between January 2013 and December 2014. All tissue samples were confirmed by pathology. This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University and conformed to the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Clinical samples were collected from patients after written informed consent was obtained.
Cells and cell culture
The GSC456, GSC4121, GSC387, and GSC3691 were gifts from UCSD. GSCs were cultured in DMEM/F12 medium with B27 and bFGF and EGF (20 ng ml−1 each).
All tissues were paraffin embedded and cut into 8-10 μm slices. After deparaffinization and antigen restoration, the primary antibody was diluted in BSA and applied overnight at 4°C. After incubation with the secondary antibodies, diaminobenzidine was added to these tumour sections, which were then counterstained with haematoxylin to visualize nuclei. The score of the IHC staining was measured according to the percentage of positive cells and staining intensity. A score of 0-6 was considered low expression, while a score of 7-12 was considered high expression.
Tumours and cells were lysed by RIPA with protease inhibitor. After quantification, equal amounts of proteins were subjected to SDS-PAGE, and after electrophoresis, the proteins were transferred to a transmembrane and blocked. Primary antibody was added overnight at 4°C. After washing, secondary antibody was applied, and the signals were visualized by chemiluminescence.
Tissues and cells were lysed with TRIzol reagent following the manufacturer’s instructions. After reverse-transcription, the DNA was then amplified with specific primers. For Circ-SMO, the sequence was described as previously.
Cells with the indicated modifications were subjected to apoptosis assays following the manufacturer’s instructions. Cells were stained, and the apoptosis rate was detected.
Plasmid construction and stable cell establishment
Junction-specific shRNAs and overexpression plasmids were described previously. After harvesting the lentivirus, cells were transduced with lentivirus using polybrene (8 mg ml−1). After incubation for 3 days, a stable cell line was successfully established.
Cells were fixed and blocked and then incubated with primary antibodies overnight. After washing 3 times, a secondary antibody was applied, and DAPI was used for nuclear counterstaining. Images were taken using an Olympus FV1000 microscope.
Certain numbers of cells were seeded into the plate and incubated with cytotoxic T cells. CCK-8 was added to the plate, and the absorption was measured at 450 nm.
LDH detecting assay
The LDH level in the medium was measured using ELISA kits following the manufacturer’s instructions. The LDH level was then normalized to the control.
Mice were randomly assigned to experimental groups for all experiments. For the animal survival analysis, mice were intracranially injected with 2,000 GSCs in 5 μl PBS and maintained until pathological symptoms from tumour burden developed or 70 days after injection. Tumour volume was then calculated.