Antibodies and reagents
Antibodies for Western blotting against phospho-Stat3 (#4113), Stat3 (#12640), Phospho-p44/42 MAPK (ERK1/2) (#9106), p44/p42 MAPK (ERK1/2) (#9102), Phospho-SHP-1 (#8849), Phospho-SHP-2 (#5431), HIF-1α (#14179) and β-actin (#58169) were bought from Cell Signaling. Peroxidase-conjugated goat anti-rabbit IgG (#111-035-003) or goat anti-mouse IgG (#115-005-003) were bought from Jackson ImmunoResearch. Antibodies for flow cytometry, Alexa Fluor 647-labeled anti-human perforin (#563576) was purchased from BD Biosciences. FITC-labeled Annexin V (#640945), PE-labeled anti-human IFN-γ (#506506), anti-human/mouse granzyme B (#372207), APC-labeled anti-human NKp46 (#137607), anti-human NKp30 (#325209), anti-human NKG2D (#320808), anti-human CD2 (#300214), anti-human CD107a (#12-1079-42) antibodies, were purchased from Biolegend. Sytox® Green Dead Cell Stain was bought from Molecular Probes (#S34860). STAT3 inhibitor Cryptotanshinone was bought from Selleck Chemicals (#35825-57-1). SHP-1 inhibitor TPI-1 (#HY-100463), SHP-2 inhibitor SHP-099 (#HY-100388) and ERK inhibitor U0126 (#HY-12031) were bought from MedChemExpress.
The NK cell line KHYG-1 was cultured in RPMI-1640 (BasalMedium, #L210KJ) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, #04-001-1ACS), 10 ng/mL human IL-2 (PeproTech, #200-02), 2 mM L-glutamine, 100U/mL penicillin and 100 μg/mL streptomycin. The NK cell line NK92 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (BasalMedium, #L110KJ) supplemented with 10% FBS, 10% horse serum, 10 ng/mL IL-2, 2 mM glutamine, 100U/mL penicillin and 100 μg/mL streptomycin. The human multiple myeloma cell line MM.1S and leukemia cell line K562 were grown in RPMI-1640 supplemented with 10% FBS, 2 mM glutamine, 100U/mL penicillin and 100 μg/mL streptomycin. All cells were maintained at 37℃ in humidified atmosphere containing 5% CO2. Normoxic or hypoxic cell culture conditions were obtained by culturing cells in a sealed incubator flushed with the mixture of 20% O2, 5% CO2, and 75% N2, or the mixture of 1% O2, 5% CO2, and 94% N2, respectively.
The expression of NK cell cytotoxicity effector molecules and KARs was analyzed by flow cytometry. For membrane staining, 5×105 cells were collected and washed with staining buffer (PBS containing 0.1% NaN3 and 0.1% BSA) three times. The cells were then incubated for 30 min on ice, according to the instructions provided with the respective antibodies. After washing 3 times with cell stain buffer, the cells were resuspended in 300 µL staining buffer in the presence of Sytox Green or 7-AAD, which were used to gate out dead cells. Acquisition of 10,000 cells per reaction was performed using a CytoFLEX Cytometer (Beckman Coulter Life Sciences). Data were analyzed with Flowjo v7.6.2 (Tree Star). For intracellular staining, 5×105 cells were collected and fixed with 1 mL 1% paraformaldehyde in PBS for 15 minutes at room temperature. After washing 3 times with cell stain buffer, the fixed cells were then resuspended in 2 mL permeabilization buffer (0.1% saponin in cell staining buffer) and incubated for 30 min at room temperature. The cells were collected again by centrifugation, stained with antibody at an optimal working concentration in permeabilization buffer for 15 min on ice. After washing three times with permeabilization buffer, the cells were resuspended cells in 300 uL cell staining buffer for final flow cytometric analysis.
CD107a degranulation assay
Degranulation of cytotoxic contents from NK cells was measured by analysis of the degranulation marker CD107a by flow cytometry. Briefly, NK cells and tumor cells were individually pre-incubated for 14-16 h at 20% or 0% O2 and after that combined in different effector-to-target (E:T) ratios at either 20% or 0% O2 for 4 h. APC labeled anti-CD107a was added to the wells within 5-10 minutes after combining NK and tumor cells. As a positive control, PMA (100 ng/mL, Sigma-Aldrich) and ionomycin (1 mg/mL; Sigma-Aldrich) were added to the NK cells during the 4 h degranulation assay.
Flow cytometric cytotoxicity assay
NK cells and tumor cells were individually pre-incubated for 24 h at 20% or 1% O2 first. NK and target cells were then coincubated under comparable conditions in different E:T ratios in a 24 well plate. After the 4 h incubation at 37°C and 5% CO2, samples were labeled with CD2-APC to distinguish effector from target cells. Cell death was detected with Annexin V-FITC and Sytox Green and analyzed on a flow cytometer. A minimum of 10,000 target events were collected per sample and the results were analyzed using Flowjo v7.6.2.
For western blotting, treated and untreated KHYG-1 and NK92 cells were lysed in buffer containing 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and protease inhibitors on ice for 30 min. Lysates were centrifuged at 12,000 rpm for 15 min and supernatants were collected. Protein concentration was determined by the BCA protein assay kit (HEART Biotech, #WB003). Equal amounts of protein were loaded and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and transferred onto a PVDF membrane (Millipore, #IPVH00010). After blocking for 1 h with 5% non-fat milk in PBS with 0.1% Tween-20 at room temperature, the membrane was incubated with primary antibody at 4℃ overnight. Immunoblots were visualized using HRP-conjugated secondary antibodies and the ECL Western Blot Detection kit (Phygene Life Sciences, #PH0353).
siRNA-mediated gene silencing in NK cells
KHYG-1 cells were kept in the above-mentioned condition. Before siRNA transfection cells were washed in pre-warmed Opti-MEM medium (Life Technologies, Carlsbad, CA, USA) and resuspended in the same medium. 106 cells were electroporated with 2 μg of siRNA in 100 μL Opti-MEM medium in 0.2 cm cuvettes with an electroporator CUY21EDIT (BEX Co. Ltd, Japan). The electroporation program was set as follows: PpV=200V, Pp on 10 ms, Pp off 10 ms, PdV=25V, Pd on 50 ms, Pd off 50 ms; Pd N=10, capacity=940 μF, and exponential decay wave type. Following electroporation, cells were resuspended in 2 mL complete media. 16-24 h after electroporation, the cells were used for western blotting or killing assay. Transfection efficiency and viability were analyzed by flow cytometry 2-6 h after electroporation, to quantitatively measure the expression of fluorescein isothiocyanate (FITC)-labeled siRNA and 7-AAD. SHP-1 mRNA was silenced by using a gene-specific siRNA pool from GenePharma (see Supplementary Table 1).
Statistical analyses were performed using the Prism software package 5.0 (GraphPad Software, San Diego, CA, USA). Data are expressed as the mean ± SEM of at least three independent experiments. Statistical significance was evaluated by two-tailed paired Student's t-test. A p < 0.05 (*), < 0.01(**), or < 0.001(***) was considered statistically significant.