Cells culture and reagents
HNC cell lines including FaDu (human hypopharynx cancer cells; ATCC®, HTB-43™), and SNU1041 (human hypopharynx cancer; KCLB, 01041.1) were obtained from the Korean Cell Line Bank. MSKQLL1 and SCCQLL1 cells were kindly provided by Prof. Se-Heon Kim (Yonsei University, Korea). FaDu, HN3, HN6, and SCCQLL1 cells were cultured in MEM (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 1% antibiotics (Gibco, 15240-062), and 1% sodium pyruvate (Gibco, 11360-070). SNU1041 cells were cultured in RPMI1640 (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), and 1% antibiotics (Gibco, 15240-062). MSKQLL1 cells were cultured in DMEM/F12 (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% antibiotics (Gibco, 15240-062). 293T cells and fibroblasts were cultured in DMEM (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 1% antibiotics (Gibco, 15240-062). Cells were incubated in 37°C and 5% CO2 under humidified conditions. Recombinant human TNF-α was purchased from PeproTech (Rockville, MD, USA). The Pan-caspase inhibitor zVAD-fmk, RIP1 inhibitor Necrostain-1 (Nec-1), RIP3 inhibitor (GSK872), and MLKL inhibitor (NSA) were purchased from Selleck (Houston, TX, USA). N-acetyl cysteine (NAC) (A9165), MnTBAP (475870), sodium nitrate (7631-99-4), sodium nitrite (7632-00-0) were purchased from Sigma-Aldrich. Cycloheximide (CST, #2112) was purchased from Signaling Technology (USA). Antibodies against RIP1 (Cell Signaling Technology Cat# 3493), p-RIP1 (Thermo Fisher Cat# PA5-105640), RIP3 (Cell Signaling Technology Cat# 13526), p-RIP3 (Cell Signaling Technology Cat# 93654, RRID: AB_2800206), MLKL (Cell Signaling Technology Cat# 91689), cleaved caspase 3 (Cell Signaling Technology Cat# 9661), cleaved caspase 8 (Cell Signaling Technology Cat# 9496), cleaved caspase 9 (Cell Signaling Technology Cat# 9505), GAPDH (Cell Signaling Technology Cat# 2118), MAVS (Cell Signaling Technology Cat# 3993), NF-kB (Cell Signaling Technology Cat# 8242), and p-NF-kB (Cell Signaling Technology Cat# 3033) were purchased from Cell Signaling Technology (USA). Anti-phospho-MLKL (Abcam Cat# ab187091) was purchased from Abcam (Cambridge, UK).
N2/Ar plasma characteristics and preparation of liquid plasma
In this study, as reported in previous experiments, a spray-type NTP system was developed and manufactured by adding Ar to N2 plasma using an N2/Ar plasma machine. The N2 gas of the machine was maintained at a flow rate of 200 sccm and the Ar gas was maintained at a flow rate of 1500 sccm. We treated N2/Ar plasma in culture medium (MEM, DMEM, or RPMI 1640) according to the manufacturer's protocol for 120 s per mL of medium (2 cm). After removing the culture medium, cells were treated with 100 µL for 96 well plates, 1mL for 12 well plates, and 3mL for 60 pi dishes.
Cell counting
Cells were treated with liquid plasma for 0, 24, and 36 h and then counted after 2 or 5 min per mL. Each cultured cell line was washed with 1× PBS, then cells were removed with 1× 0.25% Trypsin-EDTA (Gibco, 25200-056) and centrifuged at 1300 rpm for 3 min. Trypsin was then removed and the cells were resuspended in 1 mL 1× PBS. Cell solution(10µL) and Trypan Blue Stain (0.4%, 10µL) (Gibco, 15250-061) were mixed to count the dyed cells using a hemocytometer.
Cell viability assay
Cells were cultured in 96 well plates at a density of 5 × 103 cells/well. After 12 h, the plasma was replaced by medium, and the viability was calculated using Cell Counting Kit-8 (CCK8) (Dojindo, NX653) with the absorbance of the sample at 450 nm.
3D spheroid culture
Cells were added to a round 96 well plate at with 1× 104 cells/well. Following centrifugation at 3000 rpm for 15 min at 37℃, the cells were incubated at 37°C and 5% CO2 under humidified conditions.
TUNEL assay
To determine cell apoptosis, The TUNEL Assay kit (In Situ Cell Death Detection Kit, POD) (Sigma-Aldrich, 11684817910) based on deoxynucleotidyl transferase-mediated dUTP Nick end labeling (TUNEL) assay was used according to the manufacturer’s protocol. Nuclear staining was performed by diluting Hoechst (Thermo Fisher 33342) to 1:10000.
Western blotting analysis
Cells were lysed in RIPA buffer (Invitrogen, Cat IBS-BR002) containing 50 mM Tris-HCl, pH7.5, 150 mM sodium chloride, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, a protease inhibitor cocktail, and PhoSTOP (Roche Molecular Biochemicals, Bas). Protein lysate (20 µg) was separated using SDS-PAGE (10% gel) and transferred to PVDF membranes. After transfer, each membrane was blocked with 5% skim milk for 2h at room temperature and washed three times with 0.1% TBST. Following the washes, cells were incubated overnight with the primary antibody (1:1000) at 4℃ and washed three times with 0.1% TBST. The membranes were incubated with a secondary antibody (1:5000) for 2h at room temperature. Protein bands were visualized using ECL reagents (GE Healthcare Life Sciences, RPN2235) and detected using an ImageQuantTM LAS 4000 (Fuji Film, Tokyo, Japan).
Induction and inhibition of necroptosis
TNF-α, CHX, and zVAD-fmk were used to induce necroptosis in HNSCC cells, particularly in cells pretreated for 1 h before treatment. After treatment, 30 µg/mL TNF-α and 300 µM CHX (1:300) were used. To inhibit necroptosis, inhibitors, such as Nec-1 (50 µM), GSK'872 (10 µM), and NSA (2 µM) were mixed with plasma or TCZ. In addition, to prevent necroptosis by plasma, NAC, a ROS scavenger, and MnTBAP, a reactive nitrogen species (RNS) scavenger, were treated with plasma.
Immunocytochemistry microscopy
Cells were cultured on cover slips at 80% confluence. After 12 h of liquid plasma treatment, cells were washed with 1 × PBS. The cells were then fixed with 4% paraformaldehyde for 1 h and washed again with 1 × PBS. 10% BSA was added with 0.1% Triton X-100 (Sigma-Aldrich, 93443), and the cells were blocked for 1 h. The primary antibody was added to this solution, sprinkled on a cover slip, and incubated overnight at 4 ℃. After washing with 1× PBS, the secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit (A11034)) was added and the solution incubated at room temperature for 1 h. After washing three times with 1× PBS, the nuclei were stained using Hoechst and observed under a microscope.
RNA interference analysis
Using Lipofectamin RNAi MAX (Thermo Fisher, 13778150), 500 pmol siRNA was transfected into the cells. siRNAs targeting RIP3 and MLKL were synthesized by BIONEER (Daejeon, Korea). siRNA sequences of MLKL and RIP3 are as follows: human RIP3, 5` - CAG CAA UAG GAG AUU UUC U – 3` and 5` - AGA AAA UCU CCU AUU GCU G – 3`; human MLKL, 5` - GAC GUG AAC AGGAAG CUG A – 3` and 5` - UCA GCU UCC UGU UCA CGU C – 3`.
JC-1, MitoRed, and MitoSOX staining.
Cells were incubated for 24 h at 1×105 cells/well in a 12 well plate and then treated with liquid plasma for 6 h. The culture medium was removed, and the cells were washed with 1×PBS. JC-1 (JC-1; Thermo Fisher, D-1168), MitoRed (Sigma-Aldrich, 53271), and MitoSOX (MitoSOX; Thermo Fisher, M36008) were diluted 1:1000 in fresh culture medium and incubated at 37℃ for 30 min. After removing the culture medium, cells were washed with 1×PBS. Again, 1×PBS was added and living cells were observed under a fluorescence microscope (EVOS FL Auto, Thermo Fisher, Waltham, Massachusetts, USA).
ELISA
FaDu, SCCQLL1, and SNU1041 cells were seeded into 6 well plates and plasma treated for 12 h. After treatment, the supernatants were collected and centrifuged at 1300 rpm for 10 min to remove cell debris. The Human IL-6 ELISA kit (Thermo Fisher, Cat # BMS213HS) was used to detect the concentration of the indicated molecules following the manufacturer’s instructions.
Statistical analysis
Statistical comparisons between groups were carried out using the non-opposite t-test, and differences of < 0.05(*), < 0.01(**), and < 0.001(***) were considered significant. All experiments were conducted at least three times.