Ulbp1

Cells were pre-incubated with human IgG (Baxter). We purchased the following specific mAbs from Miltenyi Biotec: CD3 (IgG2a), CD14 (IgG2a), CD56 (IgG1), CD314 (NKG2D) (IgG1) and CD107a (IgG1); from BD Biosciences IFN-γ (IgG1), CD73 (IgG1), Granzyme A (IgG1), CD44 (IgG2b) and RANK (IgG1); from Beckman Coulter-Immunotech CD337 (NKp30) (IgG1), CD335 (NKp46) (IgG1) and CD336 (NKp44) (IgG1); from R&D Systems ULBP1 (IgG2a), ULBP2 (IgG2a), ULPB3 (IgG2a) anti-IL8 (IgG1), IL15α (IgG2b), CD122 (IgG1) and CD132 (IgG2a); from Ancell CD29 (IgG1), CD105 (IgG1), CD106 (IgG1) and perforin (IgG2b); from Amgen ULBP-4 (IgG1); from BioLegend CD1a (IgG1), CD90 (IgG1) and RANKL (IgG2b); from Life Technologies Granzyme B (IgG1); from Santa Cruz biotechnology Vimentin (IgG1); from LifeSpan BioSciences Cytokeratin-7 (IgG1); from Abcam Desmin (IgG). CD146 (IgG2a), M7E22 mAb anti-CD54 (IgG1), D1–12 mAb anti-HLA-DR, W6.32 mAb anti-HLA-I (IgG2a), L95 mAb anti-PVR (IgG2a), L14 mAb anti-Nectin-2 (IgG2a) and BAM195 mAb anti-MICA (IgG1) were kindly provided by Pende D. (Genoa, Italy). We purchased secondary conjugated-specific mAbs from Invitrogen or Southern Biotech. All samples were analyzed on Gallios Flow Cytometer (IL-Beckman Coulter). Data analysis was done using FlowJo software.

The expression of the NKG2D and DNAM-1 ligands on different MM cells was evaluated, after 24 h from transfection with miR-22 or miR-NC, by using fluorochrome-conjugated antibodies against MIC-A/B (Becton Dickinson, Franklin Lakes, NJ, USA), ULBP 1 (R&D Systems), ULBP 2-5-6 (R&D Systems), and CD47 (Becton Dickinson) according to the producer’s guidelines. NK cell degranulation was evaluated using CD107a staining. Specifically, miR-22- or miR-NC-transfected MM cell lines were washed twice in complete medium and incubated with PBMCs in an effector–target ratio of 10:1 in a U-bottom 96-well plate in complete medium at 37 °C and 5% CO₂ in the presence of anti-CD107a/PE (Becton Dickinson) for 3 h. Cells were then stained with anti-CD3/PcP and anti-CD56/APC to identify NK cell population. NK cells positive for CD107a were considered as degranulating/activated cells able to induce cytotoxicity, as previously described [25 (link)]. All experiments were acquired by an ATTUNE Nxt (Thermo Fisher Scientific, Waltham, Massachusetts, USA) flow cytometer. For each sample, at least 1 × 10⁴ events in the gate of interest were acquired.

A549, HCT-116, and HepG2 cells were seeded in 60 mm plates for 24 h and then treated with a predetermined decitabine dose for 24 h at 37 °C in a humidified incubator with 5% CO₂. Cells were harvested by trypsinization and washed twice with cold PBS. They were then immunostained with anti-mouse MICA, MICB, ULBP1, ULBP 2/5/6 and ULBP3 phycoerythrin (PE) (R&D Systems, Minneapolis, MN, USA), and anti-mouse HLA-A,B,C fluorescein isothiocyanate (FITC) (Beckman Coulter, CA, USA) at 4 °C for 30 minutes. Flow cytometric analysis was performed with a FC500 flow cytometer (Beckman Coulter).

To phenotype cervical cancer cell lines, cell suspensions in PBS supplemented with 0.1 % BSA and 0.02 % NaN₃ (FACS buffer) were stained for 30 min at 4 °C using antibodies to HLA-ABC (clone w6/32, Immunotools) (labeled with FITC), HLA-E (clone 3D12HLA-E, eBioscience), HLA-G (clone 87G, Biolegend), EGFR (clone EGFR.1, BD Biosciences), PVR (clone SK11.4, Biolegend), MICA/B (clone 6D4, Biolegend), ULBP2/5/6 (clone #165903, R&D systems), ULBP1 (clone #170818, R&D systems) and ULBP3 (clone #166510, R&D systems) (all labeled with PE). IgG₁, IgG_2a and IgG_2b isotype antibodies were used as negative controls. After incubation, the cells were washed with FACS buffer and analyzed using a flow cytometer LSR Fortessa (BD Biosciences). Phenotypic analyses were obtained from at least two independent experiments performed on each cell line. Data were analyzed using Kaluza software (Beckman coulter) and calculated as specific (geometric) mean fluorescence intensity (MFI) (MFI; geometric mean fluorescence of marker − geometric mean fluorescence of isotype).