Public data analysis
PIMREG mRNA expression data in primary solid tumors (TP) and adjacent normal tissues (NT) were downloaded from The Cancer Genome Atlas (TCGA) database using the Firebrowse portal 44. Glioma patient clinical information, RNAseq-based mRNA expression and mutation data were downloaded from TCGA using cBioPortal for Cancer Genomics 45. The datasets used for the analysis were Glioblastoma Multiform (TCGA, Firehose Legacy, previously known as provisional 604 samples) and Brain Lower Grade Glioma (TCGA, Firehose Legacy, previously known as provisional 530 samples) downloaded as October 2017 and TCGA-GBMLGG merged cohort 6. PIMREG expression was analyzed in glioma patients stratified according to WHO grades (II, III and IV) and mutational status of recurrently affected genes. For survival analysis, expression data was retrieved through SurvExpress platform 46, from GBMLGG (TCGA Gliomas - June 2016 n = 660) and three additional microarray-based datasets, GEO accession: GSE4412 27, GSE16011 28 and GSE4271 29.
Gene ontology was assessed with DAVID 47 and pathway enrichment with KEGG. Graphs were plotted with GraphPad Prism 4.0 software.
Glioma cell lines
The GBM cell lines U87MG, U138MG, U251MG and T98G were obtained from the American Type Culture Collection (ATCC, Manassas, USA). The GBM explant derivative U343MG cells were obtained from Dr. Carlos Gilberto Carlotti Junior’s laboratory (FMRP-USP). SF188 GBM cells and the lower-grade astrocytoma cells R186 (grade I), R259 (grade I) R286 (grade I) and UW467 (grade III) were provided by Dr. Michael Bobola (Washington University). The non-tumor astrocytes ACBRI371 were from Cell Systems (Cat# ACBRI-371) 48 and kindly provided by Dr. Elza Tiemi Sakamoto Hojo (FFCLRP-USP). All cell lines were grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C and 5% CO2.
All cell lines were tested for mycoplasm with the MycoAlert PLUS Mycoplasma Detection Kit (Lonza). U87MG and T98G were authenticated by STR matching analysis using the PowerPlex® 16 HS system (Promega, Madison, WI, USA) and the ABI 3500 Sequence Detector System (Applied Biosystems, Foster City, CA, USA).
PIMREG HA-tagged cDNA was excised with SfiI restriction enzyme from the pSfiExp-CATS-HA construct 23 and subcloned into the Doxycycline-inducible (Dox) episomal vector pRTS-1 (svh) 49, digested with the same enzyme.
For conditional PIMREG knockdown, siRNA sequences targeting the 3’ untranslated region (UTR) of the endogenous PIMREG mRNA embedded in the modified murine miR-155 structure were cloned into the BpiI site, downstream the eGFP open reading frame (ORF) of the subcloning pMIRTOP vector 50. A fragment with the miRNA-embedded siRNA sequences (miPIMREG#1: 5'-tgctgaatagaaggctcaaggtcaaggttttggccactgactgaccttgacctagccttctatt-3' and miPIMREG#2: 5'-tgctgataaataggtacccgtgagccgttttggccactgactgacggctcacgtacctatttat-3') and the eGFP ORF were excised from the pMIRTOP plasmids with Eco47II and BglII restriction enzymes and the insert was subcloned into the BglII and SwaI digested pRTS-1 (svp) vector. pRTS constructs expressing luciferase ORF (pRTS-LUC) and the miRNA-embedded siRNA targeting luciferase (pRTS-miLUC), used as controls were previously described 50.
The generated pRTS-PIMREG construct contains the hygromycin B resistance gene (hyg), whereas the pRTS-miPIMREG#1 and -miPIMREG#2 contains the puromycin gene for selection in mammalian cells. For a detailed description of the constructs contact the authors.
Transfection of U87MG and T98G cells
1x105 U87MG and T98G cells were seeded at 6-well plates and transfected with 5 μg of plasmid DNA, Lipofectamine 3000 and P3000 reagent according to manufactures’ instructions (Thermofisher). Stably transfected cell cultures were generated within approximately 7 days by hygromycin-B (200 µg/mL) and puromycin (1 µg/ml) selection of the overexpressing (pRTS-PIMREG and pRTS-LUC) and knockdown (pRTS-miPIMREG#1, pRTS-miPIMREG#2 and pRTS-miLUC) constructs, respectively. Cell populations were treated with doxycycline (0,5 µg/mL) for 2 days for conditional expression of the ectopic gene (PIMREG and LUC) or miRNA-embedded siRNA sequences (miPIMREG#1, miPIMREG#2 and miLUC).
Cell treatment with genotoxic agents and kinase inhibitor
Treatment with TMZ was carried out for 24 hours at concentrations of 25, 50, 100, 200, 400 and 600 µM for western blot analysis and at 400 µM for cell cycle and immunofluorescence experiments. For clonogenic assay, the cells were previously treated with 6.25, 12.5, 25 and 50 µM TMZ for 24 hours. IR treatment was performed at doses of 1, 2, 3, 4 and 7 Gy for western blot analysis and at 4 Gy for cell cycle assays. Cells were treated with hydroxyurea for 24 hours at concentrations of 3.75, 7.5, 15, 30 and 60 µg/mL for western blot analysis and at 15 μg/mL for immunofluorescence assay. Cells were treated with cisplatin for 24 hours at concentrations of 0.25, 0.5, 1, 2 and 4 µg/mL for western blot analysis and at 1 μg/mL for immunofluorescence assay. For inhibition of ATM and ATR pathways cells were treated for 3 hours with 10 μM of ATM (KU55933) and ATR (VE-821) kinase inhibitors prior to 400 μM TMZ treatment for 24 hours and cell lysis.
Total RNA was isolated using the RNeasy RNA extraction Kit (Qiagen) following the manufacturer's instructions. DNAse I treated RNA was reverse transcribed with random primers and High Capacity Kit (Applied Biosystems). Reactions were carried out with Power SYBR Green PCR Master Mix, according to the manufactures’ instructions (Applied Biosystems). The plates were run and analyzed by 7500 RealTime PCR System (Applied Biosystems). Primer sequences are available upon request. HPRT was used as endogenous control and relative gene expression was calculated using the 2−ΔΔCT equation 51. Statistical analyzes were performed with the One-way ANOVA test, using the GraphPad Prism 4.0 software.
Immunobloting and immunofluorescence
Cells were lysed in RIPA buffer with protease inhibitor PMSF. Protein extracts were separated by electrophoresis on 6 or 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Hybon-ECL; Amersham Biosciences, Arlington, IL). The membranes were blocked and probed with specific antibody, followed by incubation with secondary antibody conjugated to horseradish peroxidase (HRP). Protein signals were detected by chemiluminescence and the images were captured by ChemiDoc™ Imaging System (Bio-Rad). Primary antibodies were: anti-PIMREG (anti-CATS 2C4 (1:10) , anti-β-Actin (Cell Signaling, #3700) (1:1000), anti-phospho-H2AX(S139) (γH2AX; Abcam, ab26350) (1:1000), anti-phospho-ATM(S1981) (Abcam, ab81292) (1:1000), anti-ATM (Abcam, ab32420) (1:1000), anti-phospho-ATR(S428) (Cell Signaling, #2853) (1:1000), anti-ATR (Cell Signaling, #2790) (1:1000), anti-phospho-RPA32/RPA2 (S33) (Abcam, ab87278) (1:200), anti-RPA32/RPA2 (Abcam, ab2175) (1:200), anti-Cyclin A (Santa Cruz, sc-271645) (1:500) and anti-Cyclin B (Santa Cruz, sc-7393) (1:500).
Secondary antibodies were purchased from Cell Signaling: anti-mouse (#7076) (1:5000), anti-rat (#7077) (1:2000) and anti-rabbit (#7074) (1:5000). Densitometric quantification of the protein bands was performed with ImageJ v1.38. Statistical analyzes were performed with the One-way ANOVA test, using the GraphPad Prism 4.0 software.
For immunofluorescence, U87MG cells were grown on coverslips and treated with genotoxic agents for 24 hours. After that cells were ﬁxed with 4% paraformaldehyde (PFA) for 15 minutes, permeabilized with PBS/0.25% Triton X-100 for 15 minutes. Unspecific binding was blocked with PBS/5% BSA for 1 hour. Coverslips were incubated with anti-PIMREG (CATS 2C4) mAb diluted 1:2, anti-Cyclin A (sc-271645) diluted 1:200 and anti-phospho-H2AX(S139) (Merck, #05-636) diluted 1:500 at 4ºC overnight in a humidified chamber. Secondary Alexa Fluor 488 (ThermoFisher, A-11006) and Alexa Fluor 594 (ThermoFisher, A-11005) antibodies diluted 1:500, were used for detection of the primary antibodies for 1 hour at RT. Nuclei were counterstained with DAPI (ThermoFisher, 62247) and cells were mounted on slides using ProLong™ Gold Antifade Mountant (ThermoFisher, P10144). Images were captured by Leica DMI6000B ﬂuorescence microscope. Statistical analyzes were performed with the Student’s T-test, using the GraphPad Prism 4.0 software.
Cell cycle analysis
Cell cycle analysis was performed by DNA content quantification. Cells were washed with phosphate-buffered saline (PBS) with 5 mM EDTA, fixed in 70% ethanol for 30 minutes on ice, washed with PBS-EDTA and stained with 20 µg/mL propidium iodide (PI) containing 10 µg/mL RNAse A for 30 minutes at 37°C. Cell cycle was analyzed using FACSCanto (BD Biosciences) and ModFit LT 3.3 software. Statistical analyzes were performed with the Student’s T-test, using the GraphPad Prism 4.0 software.
Stably transfected U87MG and T98G cells expressing of the ectopic gene (PIMREG and LUC) or miRNA-embedded siRNA sequences (miPIMREG#1, miPIMREG#2 and miLUC) were seeded in 96-well plates at density of 500 cells/well. At the indicated time points, cells were washed, fixed in 70% ethanol for 10 minutes and stained with 0.5% crystal violet solution for 15 minutes. After that, cells were washed and incubated with 10% acetic acid solution for 30 minutes. Proliferation was evaluated by measuring the absorbance at 540 nm with an Elisa microplate reader. Statistical analyzes were performed with the One-way ANOVA test, using the GraphPad Prism 4.0 software.
For apoptosis evaluation, cells were seeded in 12-well plates at density of 5x104 cells/well. After 24 hours of incubation under normal culture condition, FBS was removed and cells were incubated further for 144 hours. After that, cells were washed in ice-cold PBS and stained with annexin V for 15 minutes at RT. PI was added (2 ng/µL) prior to the FACS analysis, performed using FACSCanto and FACSDIVa software (BD Biosciences). Statistical analyzes were performed with the One-way ANOVA test, using the GraphPad Prism 4.0 software.
Clonogenic survival assay
Stably transfected U87MG and T98G cells expressing of the ectopic gene (PIMREG and LUC) or miRNA-embedded siRNA sequences (miPIMREG#1, miPIMREG#2 and miLUC) were seeded in 6-well plates at density of 500-2500 cells/well and treated with TMZ. The drug was removed and cells were further incubated for 9 days under normal culture conditions. After that period, cells were fixed with 4% PFA, stained with 0.5% crystal violet and the number of colonies formed were counted. Statistical analyzes were performed with the One-way ANOVA test, using the GraphPad Prism 4.0 software.
Migration of U87MG and T98G cells expressing of the ectopic gene (PIMREG and LUC) or miRNA-embedded siRNA sequences (miPIMREG#1, miPIMREG#2 and miLUC) was assessed by two independent methods.
For the wound healing assay, cells were grown to confluence and wounded by making a scratch with a sterile pipette tip. Images were acquired at 0, 72 and 144 hours following the scratch using an inverted bright field microscope. ImageJ v1.38 software was used to calculate the percentage of wound closure determined by reduction of the distance between the wound edges from time 0 set as 100%. Statistical analyzes were performed with the One-way ANOVA test, using the GraphPad Prism 4.0 software.
Transwell migration experiments were performed in 8 μm pore-sized transwell plates (Greiner Bio-One). Cells were seeded above the filter at a density of 3x104 cells/well in 200 μl of serum-free medium and the lower compartment was filled with and 500 μL of 10% FBS containing medium. After 24 hours, the cells which migrated through the filter and reached the lower compartment were fixed with 4% PFA and stained with crystal violet 0.1%. The migrated cells were counted using an inverted microscope. Statistical analyzes were performed with the One-way ANOVA test, using the GraphPad Prism 4.0 software.