T98G is a fibroblast-like cell line that was isolated from the brain of a glioblastoma multiforme white, 61-year-old, male patient. U87MG is a cell line with epithelial morphology that was isolated from malignant gliomas (glioblastoma) from a male patient. Both cell lines were cultured in Dulbecco’s modified Eagle’s medium–high glucose (DMEM) (Sigma Cat no. D6429-500 mL) supplemented with 10% FBS (fetal bovine serum), 1% penicillin–streptomycin at 37 °C in a 5% CO2 environment. When cells reached 80% of confluence, they were split and sub-cultured at a concentration of 10.000 cells/well in 96-well plates for experimental procedures.

U87MG and T98 cells were treated for 72 h with ATP (ATP disodium salt by Tocris Biosciences Catalog #3245) that was dissolved in media and the pH was adjusted to 7.4 with NaOH, and with the following P2X7R antagonists: AZ10606120 (Tocris Biosciences Catalog #3323) and A74003 (Tocris Biosciences Catalog #3701).

XTT (TACS® XTT Cell Proliferation Assay Kit, R&D System, Minennapolis, MN, Cat. No. 4891–025-K) was performed, according to the manufacturer’s procedure. U87MG and T98G were plated in a 96-well plate at a density of 10,000 cells/well. XTT assay was performed at 24 h, 48 h, 72 h. Cell viability was examined by measuring the reduction of formazan salts (3-(4,5-dimethylthiazole-2-yl)−5-(4-sulfophenyl)−2H-tetrazolium MTS) contained in the kit. Living cells reduce MTS to purple formazan salts, which absorb light at 492 nm. Cell viability was assessed by measuring absorbance using a microplate photometer (Victor 4, PerkinElmer, Waltham, MA, USA) and was expressed as the percentage of cell viability in relation to the untreated controls. The absorbance values measured with a plate spectrophotometer are directly proportional to cell viability.

LDH assay (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega, Madison, WI, USA, Cat. No.: G1780) was performed, following manufacturer’s instructions. U87MG and T98G were plated and treated with the same procedure used for XTT assay and extracellular and intracellular LDH amount was read by measuring absorbance at 490 nm with a microplate photometer (Victor 4, PerkinElmer, Waltham, MA, USA). LDH assay was performed at 24 h, 48 h, 72 h. Extracellular LDH was measured in the medium released from cells, while intracellular one was measured from cell lysate. Results refer to the percentage of extracellular LDH on total LDH (where total is calculated as extracellular + intracellular content).

Bradford protein assay (Quick Start™ Bradford Protein Assay Kit, Biorad, Hercules, CA, USA) was performed to quantize protein levels. Cells were treated and lysed in 100 μL Triton X-100 (0,9%) diluted at a ratio of 1:10. In total, 10 μL of each treatment was used to calculate the amount of protein per well. BSA was chosen as standard to develop a calibration curve ranged from 1 mg/mL to 0 mg/mL and the protein amount was measured as a function of absorbance at 570 nm using a microplate photometer (Victor 4, PerkinElmer, Waltham, MA, USA). Results were expressed as a total μg of proteins per well. Bradford assay was performed at 24 h, 48 h, 72 h.

Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation using the BrdU Cell Proliferation Assay Kit purchased from Cell Signaling (Cat. No.:6813). Cells were plated at a density of 10,000 cells/well in 96-well plates. BrdU was added overnight during the 16–20 h prior to the end of the incubation. The assay was performed according to the manufacturer's instructions. The kit detects 5-bromo-2'-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation using an anti-BrdU antibody. When cells are cultured in a medium containing BrdU, this thymidine analogue is incorporated into the newly synthesized DNA of proliferating cells. After removing the medium, the cells are fixed, and the DNA is denatured using a fixation/denaturation solution. A mouse monoclonal anti-BrdU antibody is then added to detect the incorporated BrdU (DNA denaturation is necessary to improve the accessibility of the incorporated BrdU to the detection antibody). The anti-mouse IgG-HRP conjugated antibody is then used to recognize the bound detection antibody. The HRP substrate (IMB) is added to detect the color. The absorbance of the developed color is proportional to the amount of BrdU incorporated into the cells, which is a direct indication of cell proliferation. The obtained data is represented in a graph showing the percentage (%) of BrdU measured relative to the control for each tested dose.

Both U87MG and T98G were plated in T25 flask at a density of 500.000 cells and treated at 4 h and 24 h. After 4 h or 24 h, cells were scraped in PBS without Ca2+ and Mg2+ and centrifuged at 1100 rpm for 5 min. Then, cells were lysed in RIPA buffer [1 mM EDTA (Cat. No.: E7889), 150 mM NaCl (Cat. No.: S9888), 1% igepal (Cat. No.: I3021), 0.5% sodium deoxycholate (Cat. No.: D-6750), 50 mM Tris–HCl, pH 8.0 (Cat.No.: T-3038) (Sigma-Aldrich, St. Louis, MO, USA), and 0.1% sodium dodecyl sulphate, SDS, (Cat. No.:1610416—Bio-Rad, Hercules, CA, USA)] containing protease inhibitor cocktail diluted 1:250 (Cat. No.: P8340—Sigma–Aldrich, St. Louis, MO, USA). The total protein amount was measured following the Bradford protein assay, as described above. Precast BIS–TRIS polyacrylamide gels with a 4–12% polyacrylamide gradient (Invitrogen, Carlsbad, CA, USA) were used for the experiments. 30 μg of proteins/sample were mixed with 4 × Bolt™ LDS Sample Buffer (Cat. No.: B0007—Novex, Carlsbad, CA, USA) and 10 × Bolt™ Sample Reducing Agent (Cat. No.: B0009—Novex, Carlsbad, CA, USA), boiled for 5 min at 95 °C and, eventually, undergone to the electrophoresis. Then, proteins were transferred to a PVDF membrane (Invitrogen, Carlsbad, CA, USA) using iBlot™ 2 Gel Transfer Device (Invitrogen, Carlsbad, CA, USA) and different antibodies were tested. Primary antibodies were all prepared in Flex Solution (iBind™ Flex Solution Kit, Invitrogen, Carlsbad, CA, USA) and were incubated overnight at 4 °C with gentle shaking; except for β-actin and pCREB antibodies, which were incubated for 2 h. The day after, the primary antibody was removed, and the membrane was washed three times with TBS-T. After that, the membrane was incubated for 1 h with the secondary antibody, dissolved in Flex Solution (iBind™ Flex Solution Kit, Invitrogen, Carlsbad, CA, USA). Following three washes in TBST, bands were detected by chemiluminescence (ChemiDoc™ XRS, Biorad, Hercules, CA, USA), rinsing the membrane with ECL reagents (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Thermo Scientific™, Rockford, IL, USA, and Pierce™ ECL Western Blotting Substrate).

Antibody information used for Western immunoblotting analysis:

Antibody

Diluition

Producer

Catalog Number

β-actina

1:1000

Sigma-Aldrich

#A 5316

pCREB (ser133)

1:1000

ThermoFisher

PA1-4619

Total CREB

1:500

ThermoFisher

PA1-850

P21

1:500

Cell signaling

#64016

LC3A

1:250

Novus Biologicals

NB100-2331

Beclin 1

1:1000

Novus Biologicals

NB500-249

Cleaved caspase 3 (Asp 175)

1:1000

Cell signaling

#9661

pSTAT 3

1:500

Cell signaling

#9145

totSTAT 3

1:1000

Cell signaling

NFKB

1:1000

Cell signaling

#8242

GAPDH

1:1000

Cell signaling

#2118

Anti-mouse

1:3000

Sigma-Aldrich

#A 3682

Anti-rabbit

1:15000

JacksonImmuno

#111–035–045

According to manufacturer's instructions, the Human Interleuchin 6 (IL 6) levels in the culture media were assayed by enzyme-linked immunosorbent assay (Human IL-6 DuoSet ELISA, Cat No: DY206-05, R&D Systems). Cells were plated at a density of 10,000 cells/well in 96-well plates. The day before the dosage, the wells were coated with capture antibody overnight. At the end of treatment, the culture medium was collected and then diluted 1:2 to quantify interleukin levels and the other steps were performed as instructed. The results were calculated, through a calibration curve in the range 9.4—600 pg/mL, as picograms of IL 6 normalised to milligrams of total proteins.

Total cytoplasmic RNA was extracted using the RNeasy Micro Kit (Qiagen, Hilden, Germany), which included a 15-min DNase treatment. RNA concentration was measured using the Qubit™ RNA HS Assay Kit (Thermo Fisher Scientific). Aliquots of RNA were reverse transcribed into cDNA using random hexamer primers.

Quantitative changes in mRNA levels were assessed by real-time PCR using the following cycling conditions: one denaturation cycle at 95 °C for 3 min; 40 amplification cycles at 95 °C for 5 s and 60 °C for 10 s; followed by one melting cycle at 95 °C for 1 min, 60 °C for 30 s, and 95 °C for 30 s, using the Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Stratagene, La Jolla, CA, USA). PCR reactions were performed in a final reaction volume of 20 µL using the AriaMx real-time PCR system (Stratagene).

The primers used for gene expression analysis targeted P2X7 receptor (P2X7R) isoform A, B and Bs. Relative mRNA concentrations were calculated from the take-off point of the amplification curves (threshold cycle, Ct) using the comparative quantification method provided by the Stratagene software and based on the 2^ − ΔΔCt method.

This analysis estimates the target mRNA level of a given sample relative to the mean target mRNA levels of untreated control samples (calibrator value), thereby allowing statistical analysis of deviations from the mean, including among control samples. Ct values for GAPDH expression were used as the normalization signal. In each assay, PCR efficiency was also calculated using serial dilutions of an experimental sample.

Each experiment was repeated at least three times. All the statistical analyses were performed with PrismTM computer program (GraphPad, San Diego, CA, USA Software version 7.4). Data were analyzed by a one-way ANOVA, followed by Dunnett’s test. Statistical significance was determined at α = 0.05 level. Differences were considered statistically significant when p < 0.05.