Summit 4

Cellular synchronization at the G1/S boundary was performed using thymidine (Sigma-Aldrich, St. Louis, MO, USA) following the method proposed by Chen and Deng [18 (link)]. After the cell cycle resumed, cells were collected at 0, 2, 4, 8, 16, 24, and 48 h for further analyses.
Cells seeded for the synchronization assay were collected and fixed overnight at 4 °C in 70% ice-cold ethanol. After washing with ice-cold PBS, cells were stained with a 25 μg/mL propidium iodide solution in the presence of 200 μg/mL RNase A at 37 °C for 45 min in the dark. The cell cycle distribution was determined by flow cytometry in a Beckman Coulter Gallios Flow cytometer in the General Research Services SGIker of the UPV/EHU, with a total acquisition of 10,000 events. The percentage of cells in different phases of the cell cycle was analyzed using Summit 4.3 software (Dako, Hovedstaden, Denmark).

Cells were fixed with ice-cold ethanol and re-suspended in PBS containing RNase I (100 mg/mL, Sigma) and propidium iodide (50 mg/mL, Sigma). Samples were subjected to fluorescence-activated cell sorting (FACS, Calibur, Becton Dickinson), and data were analyzed using the Summit 4.3 software (DAKO Cytomation) as previously described (Federico et al., 2016 (link)).

Cells were detached with PBS/EDTA 1 M and stained with APC-conjugated mouse anti-Human MET (3D6; BD Biosciences, Allschwil, Switzerland). For isotype control, an APC-conjugated anti-mouse Ig antibody (BD Biosciences) was used. Cells were co-stained with DAPI. MET expression was analysed by Summit 4.3 software (Dako, Santa Clara, CA, USA). The signal derived from the isotype control was set as: 0 < MFI < 10¹,and cells were considered MET positive when MFI > 10¹.

Six to eight mm mean diameter tumors from neuT and neuT-pfpKO mice were minced with scalpels and then incubated with 1 mg/mL collagenase IV (Sigma-Aldrich) in RPMI-1640 (Life Technologies, Monza, Italy) at 37 °C for 1 h in an orbital shaker. The cell suspension was washed in PBS supplemented with 2% FBS (Invitrogen), incubated in a buffer for erythrocyte lysis (155 mM NH₄Cl, 15.8 mM Na₂CO₃, 1 mM EDTA, pH 7.3) for 10 min at RT, and then washed in RPMI-1640 supplemented with 10% FBS (Invitrogen). The cell suspension was passed through a 70-µm pore cell strainer (BD Biosciences, Milano, Italy), centrifuged (1400 rpm for 10 min), and re-suspended in a buffer for erythrocyte lysis. After 10 min of incubation at RT, tumor-infiltrating leukocytes were washed with RPMI-1640 supplemented with 10% FBS (Invitrogen), centrifuged (1400 rpm for 10 min), re-suspended in PBS, treated with Fc receptor blocker (anti CD16/CD32; BD Biosciences, Milano, Italy), and stained with the following antibodies: anti-mouse CD45 VioGreen, anti-mouse CD3 FITC, anti-mouse CD49b PE, anti-mouse CD4 APC Vio770, anti-mouse CD8 VioBlue, anti-mouse γδ TCR PE/Cy7, anti-mouse CD11b FITC, anti-mouse F4/80 APC, and anti-mouse GR-1 PE (Miltenyi Biotech, Milano, Italy) [27 (link)]. Samples were acquired and analyzed on a CyAn ADP (DakoCytomation, Milano, Italy) using Summit 4.3 software (DakoCytomation, Milano, Italy).

Peripheral blood mononuclear cells (PBMCs) were isolated from 20 to 25 ml of peripheral blood samples via Ficoll density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). According to the recommendation of the international working group of the experts in flow cytometry [7 (link)], CD3, CD4, CD25, CD127, and FoxP3 markers were used to analyze human Treg cells using a gating strategy illustrated in Supplementary Figure 1. Specifically, CD3-FITC, CD45-PerCP, CD127-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD4-Alexa Fluor® 700, (EXBIO a.s., Prague, Czech Republic), Foxp3-PE, and CD25-Brilliant Violet (BioLegend, San Diego, USA) antibodies (Abs) were used. For intracellular staining, we used Foxp3-Fix/Perm and Foxp3-PErm Buffers (BioLegend, San Diego, USA). The flow cytometric data were collected on a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using Summit 4.3 software (Dako, Glostrup, Denmark).

For cell sorting, A549/pr(IFN-β).GFP cells were infected at 0.04 FPU/cell. At 8 hpi, cells were trypsinised, resuspended in Mg²⁺- and Ca²⁺-free PBS and passed through a 30 µm pore filter to obtain single-cell suspensions. Cell sorting was carried out using an Influx cell sorter (BD Bioscience) equipped with 5 solid-state lasers. eGFP was excited with 488 nm laser and collected through a 517/30 filter. A Forward Scatter versus Side Scatter plot was used for excluding debris. Data from list files of 10.000 events were analyzed using Summit 4.3 Software (DAKO). FACS analysis was performed with A549/pr(IFN-β).GFP cells trypsinised to obtain single-cell suspension and fixed in PBS/1% formaldehyde. GFP expression was examined using a BD FACScan flow cytometer and data were analyzed using FlowJo (Treestar).

TIGK cells were cultured and treated in the same conditions described in the Real-time PCR section.
TIGK cells treated with different concentrations of glucose, including both attached and disassociated, were harvested. Early apoptotic, late apoptotic, necrotic, and live cells were determined using an Annexin V Apoptosis Detection Kit APC (Thermo Fisher Scientific, Waltham, MA, USA). Harvested cells were washed with binding buffer. After centrifugation, cells were re-suspended in 100 μL of binding buffer and incubated with APC-conjugated Annexin V for 15 min at room temperature. After washing, 5 μL of propidium iodide (PI) was added. The cells were then immediately subjected to flow cytometry analysis without being fixed by any fixatives using LSR Fortessa (BD bioscience, San Jose, CA, USA) at the Flow Cytometry Core, Research Resources Center, University of Illinois Chicago. Results were analyzed using Summit 4.3 software (Dako Cytomation, Glostrup, Denmark). Cell debris and doublets were gated out in the analysis. There were 3 replicates at each culture condition and each time point.