Human specimens
In the present study, tumor and adjuvant normal brain tissues of 10 GBM surgical specimens (male=4, female=6, median age=11.8±3.7 years) were collected from Children`s Hospital of Fudan University. In detail, these GBMs consist of 5 primary (male=2, female=3, median age=12.1±3.9 years) and 5 recurrent (male=2, female=3, median age=11.7±3.1 years) tumors. All the samples were immersed in RNAlater Stabilization Solution (ThermoFisher Scientific, Cat:AM7020) and stored in -80 oC freezer. Pathological examination of all participants was independently confirmed by 3 board-certified neuropathologists according to the 2016 World Health Organization (WHO) grading classification [33]. All the protocols involved in this study have been approved by the ethics committee of the Children`s Hospital of Fudan University. All families of involved participants have signed the written informed consent.
Reagents
Antibodies of anti-LITAF(sc-166719) and anti-γH2AX (sc-517336) were purchased from Santa Cruz Biotechnology (Shanghai, China), antibodies of anti-ACTIN (#4970), anti-phos-γH2AX (Ser139, #80312), anti-STAT6 (#5397), anti-STAT3 (#9139), anti-TLR1 (#2209), anti-STAT5A (#4807), anti-IFNAR1 (sc-7391), anti-Flag (#14793), anti-phos-ERK1/2 (#8544), anti-ERK1/2 (#4695), anti-p38 (#8690), anti-phos-p38 (#4511), anti-AKT (#4691), anti-IκB-α (#4812), anti-phos-IκB-α (#2859) and HRP-linked secondary mouse (#7076)/rabbit(#7074) antibody were from Cell Signaling Technology (Danvers, USA). Florescence secondary antibodies Alexa Fluor Plus 488(#A32723)/555(#A32727) were purchased from ThermoFisher Scientific (Waltham, USA). Kavain (sc-201077) was purchased from Santa Cruz Biotechnology (Dallas, USA). DAPI (4',6-Diamidino-2-phenylindole Hydrochloride) dye (sc-3598) and EdU (2'-Deoxy-5-ethynyluridine-d1) were purchased from Santa Cruz Biotechnology (Dallas, USA). PI (Propidium Iodide, #4087) was from Cell Signaling Technology (Danvers, USA).
Cell culture
U87 (also U-87 MG, from American Type Culture Collection) cells were maintained in ATCC-formulated Eagle's Minimum Essential Medium (Cat: No. 30-2003) containing 10% FBS (Gibco, Cat: 10091148) and 50 mg/mL penicillin/streptomycin (P/S, Cat: 10378016). DK (also DK-MG, from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) cells were cultured in RPMI-1640 containing 10%FBS, 50 mg/mL P/S and 2 mM L-glutamine. T98G cells also were purchased from ATCC and cultured in the same condition with U87 cells. HEK293t cells were cultured in Dulbecco's Modified Eagle Medium (Gibco, Cat: 11965092) containing 10% FBS and 50 mg/mL P/S. All cells were maintained in an incubator with 37°C and 5% CO2 conditions.
Construct of stable cell lines
For lentivirus packaging in constructing stable cell lines, HEK293A cells were co-transfected with viral vectors (expressing shLITAF or LITAF) and packaging plasmids (psPAX.2 and pMD2.G) using polyjet reagent from Signagen (Frederick, USA) following manufacturer’s instructions. 48 hours later of transfection, lentivirus supernatant was filtered using a 0.45-mm filter for the further infection of target cells with 5 mg/ml polybrene (GM-040901B) from Genomeditech (Shanghai, China). 1 day later of infection, the medium was refreshed using a culture medium containing 1 mg/ml puromycin (A1113802) from ThermoFisher Scientific (Waltham, USA).
Quantitative Real-Time PCR (qPCR)
Total RNA was purified using the Trizol reagent (Cat: 15596026, ThermoFisher Scientific). Subsequently, 1 μg total RNA was reversely transcribed into cDNA using the PrimeScriptTM RT reagent Kit (Cat: RR036A, TaKaRa). Finally, qPCR was performed using TB Green Fast qPCR Mix (Cat: RR430A, TaKaRa) on 7500 Real-Time PCR systems (Applied Biosystems, Waltham, USA). The ΔΔCt method was used for relative quantification of target genes with the internal control of GAPDH mRNA.
Oligos for qPCR were listed as follows. LITAF: Forward, 5'-ATG TCG GTT CCA GGA CCT TAC-3', Reverse, 5'-TAC GAA GGA GGA TTC ATG CCC-3', TNF-α: Forward, 5'-CCT CTC TCT AAT CAG CCC TCTG-3', Reverse, 5'-GAG GAC CTG GGA GTA GAT GAG-3', CCL-2: Forward, 5'-CAG CCA GAT GCA ATC AAT GCC-3', Reverse, 5'-TGG AAT CCT GAA CCC ACT TCT-3', IL-1: Forward, 5'-TGG TAG TAG CAA CCA ACG GGA-3', Reverse, 5'-ACT TTG ATT GAG GGC GTC ATTC-3', CXCL-1: Forward, 5'-GCT GCT CCT GCT CCT GGT AG-3', Reverse, 5'-ACA GCC ACC AGT GAG CTT CC-3', STAT3: Forward, 5'-CAG CAG CTT GAC ACA CGG TA-3', Reverse, 5'-AAA CAC CAA AGT GGC ATG TGA-3', STAT5A: Forward, 5'-CAG TGG TTT GAC GGG GTG AT-3', Reverse, 5'-GTC GTG GGC CTG TTG CTT AT-3', GAPDH: Forward, 5'-GGA GCG AGA TCC CTC CAA AAT -3', Reverse, 5'-GGC TGT TGT CAT ACT TCT CAT GG-3'.
Western blot (WB)
Tumor tissues were lysed using tissue total protein lysis buffer (Cat: C500028-0010) from Sangon Biotech (Shanghai, China), followed by quantifying using BCA Protein Assay Kit (Cat: PC0020) from Solarbio company (Beijing, China). Cells were lysed in a home-made 1×SDS loading buffer containing 50mM Tris pH6.8, 2% SDS, 0.025% Bromophenol blue, 10% glycerol, and 5% BME. Firstly, protein samples were separated using a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose (Cat:10600002, Whatman) filter membrane. Subsequently, membranes were blocked using 5% skim milk/TBST buffer for 1 hr at room temperature. After washed with TBST, membranes were probed using primary antibodies (4oC, overnight). The next day, membranes were probed using HRP-conjugated secondary antibodies at room temperature for 1 hr. Finally, protein bands were quantified using ECL substrates and imaged using Tanon-5200Multi equipment (Shanghai, China).
Immunofluorescence (IF)
Cells were seeded in a 6-well plate with condition medium with/without Kavain (50 μg/ml, for 6 hr). After briefly washed using cold 1×PBS, cells were fixed with 4% paraformaldehyde fix solution (Cat: E672002, Sangon Biotech) for 15 min, followed by being permeabilized with 0.2% Triton X-100 (Cat: T8200, Solarbio) at room temperature for 15 min. Subsequently, cells were blocked using 5% BSA/PBS in room temperature for 30 min and then incubated with first antibodies (in 5% BSA/PBS) at 4°C overnight. The next day, cells were briefly washed using 1×PBS, and then incubated with Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (in 1×PBS) at room temperature for 1.5 hr. Finally, cells were washed using 1×PBS for 3×5 min at room temperature and then subjected to microscopy.
Immunoprecipitation (IP)
HEK293t cells transfected with indicated plasmids were lysed using cold mild lysis buffer (MLB) containing 2 mM EDTA, 20 mM Tris, 1% NP-40, 100 mM NaCl, 1 mM Na3VO4, 50 mM NaF plus protease inhibitor cocktail (Cat: HY-K0010, MedChemExpress). After quantified using BCA Protein Assay Kit (Cat: PC0020), equal cell lysates were incubated with anti-Flag antibody at 4°C for 2 hours, and subsequently with Anti-FLAG M2 magnetic beads (Cat: M8823, Merck) at 4°C for 3-5 hours. Finally, the beads were washed with cold MLB 3×10 min, eluted by 1×SDS loading buffer (~1/10 input), boiled at 100°C for 10 min, and then separated by SDS-PAGE to WB detection.
Cell growth curve
Cells were cultured in growth medium (with/without 50 μg/ml) in 96-well microplate (Cat: nos. 3798, Corning) overnight. The next day, cells were subjected to X-ray irradiation treatments (1 Gy/min) using X-RAD 320 device (Precision X-ray). After irradiation, cells were maintained in a CO2 incubator (at 37°C), and cell viability was determined at indicated time points using MTT Assay Kit (Cat: ab211091, Abcam) following manufacturer’s instruction. Each treatment was replicated at least three wells, and the relative MTT optical density (OD) by normalized by the value of 0 day.
Colony formation assay
Melted agarose (1.0% in ddH2O) was fully mixed with an equal volume of 2× DMEM medium at a temperature of 42-45°C in a sterile condition and then placed onto 3.5-cm dishes to form base agarose gel. U87 and DK cells (1000 cells/well) suspended in DMEM medium with 10% fetal bovine serum and 0.3-0.4% agarose were plated onto the prepared plates with base agarose gel. If required, cells were treated with PBS or Kavain (200 mg/ml) every 2 days. As to irradiation, cells were irradiated (4Gy, with a rate of 1.8 Gy/min) at 3, 8, 13 and 18 day post-implantation. Finally, the colonies were fixed in 4% paraformaldehyde, stained with 0.1% crystal violet and counted.
Tumor xenograft assay
BALB/C nude mice (4-5 weeks) were purchased from Shanghai SLAC laboratory animal Co. Ltd (Shanghai, China). DK and U87 cell lines were trypsinized and then resuspended with a mixture of equal volume PBS and Matrigel (107 cells/ml). The prepared cell suspensions were injected subcutaneously into both shoulders of a nude mouse. In case of Fig2, mice injected with wt and shLITAF (or oeLITAF) cells were divided into two subgroups (n=4): (1), Control (Con) group without irradiation and (2), Irradiation (IR) group receiving 5-Gy localized radiation at 5, 10 and 15 dpi (dpi, day post-injection). In case of Fig3, mice injected with U87(wt and LITAF-/-) or DK cells lines were divided into four subgroups (n=4): (1), Control group, only receiving equal volume PBS treatment, (2), IR group, receiving 5-Gy localized radiation at 5, 10 and 15 dpi, (3), Kava group, receiving an intratumoral kavain treatment (100 µl, 4 mg/kg) every 3 days, (4), IR+Kava group, receiving irradiation and kavain treatment as (2) and (3). The tumor sizes were measured every 5 days to draw a tumor growth curve. One month later, mice were sacrificed with sodium pentobarbital (50 mg/kg) via intraperitoneal (i.p.) injection. The wet weight of each tumor was measured.
Public data analysis
LITAF gene expression in gliomas tissues and LITAF-based overall survival analysis (Illumina Hiseq platform) were analyzed using a web-based tool in the Chinese Glioma Genome Atlas (CGGA, http://www.cgga.org.cn/index.jsp).LITAF protein level in (low and high) gliomas tissues are quantified using the public data from The Human Protein Atlas database.
Statistical analysis
In the present study, all the data were presented as mean ± SD with at least 3 duplications. Statistical analyses were conducted on the software of GraphPad Prism7 (San Diego, USA). For comparisons between two groups, student’s t-test was used, for comparisons between three or more groups, one- or two- way ANOVA test was used. A P value < 0.05 was considered as statistical significance.
Data availability
The data that support the findings of this study are available from the corresponding authors upon request.