*4.4. Estimation of Extracellular pH and Temperature and Number of Melanoma Cells*

RPMI-1640 serum-free media were placed in 48-well plates (1 mL: per well) and exposed to µ-DBD plasma using air as the feeder gas for 30, 60, 180, and 300 s. After exposure, the temperature and pH of the media were measured immediately in triplicate with an infrared (IR) camera (Fluke Ti100 Series Thermal Imaging Cameras, Everett, WA, USA) and pH meter (Eutech Instruments, Singapore). The G-361 cells were counted and DIC images (Leica, DMi8, Wetzlar, Germany) were taken after different CAP exposure times.

#### *4.5. Glucose Uptake*

G-361 cells after culture were treated with SN (100 nM), CAP (180 s), and a combination of SN + CAP and kept for the next 24 h in a CO<sup>2</sup> incubator. After incubation, G-361 cells were incubated with 100 µM 2-NBDG, which is a fluorescent derivative of 2-deoxy-d-glucose, for 45 min. The cells were trypsinized, collected, and washed with PBS and then analyzed by flow cytometry to collect green fluorescence. In a separate experiment, G-361 cells were fixed and permeabilized separately using 10% ethanol and 3.7% paraformaldehyde. They were then incubated with DAPI for 10 min to counterstain the nucleus, along with 2-NBDG. The fluorescence staining intensity and intercellular locations were examined using an Olympus IX83-FP confocal microscope (Tokyo, Japan).

#### *4.6. qPCR Gene Analysis (Autophagy Related and Transcriptional Factor)*

Any alterations in the cellular level are associated with changes in the gene expression at the mRNA level. To clarify this at molecular levels, we identified genes responsible for the induction of *mTOR*-mediated autophagy and related transcriptional factors and checked their levels by the production of plasma-generated reactive species. The desired primers for *mTOR* and *RAS*/*MEK*-mediated autophagy and its relevant transcriptional factors were designed and synthesized. G-361 melanoma cells were grown in Petri plates with a size of 35mm<sup>2</sup> and treated with CAP, SN, and CAP + SN, respectively, as described before. After incubation for 24 h, the cells were trypsinized and collected for RNA isolation using Trizol (Invitrogen, Carlsbad, CA, USA), after which q-PCR was performed with a Biorad 2X SYBR green mix (Biorad, Seoul, Korea). Reactions were carried out in a Biorad thermal cycler (Biorad, Seoul, Korea), and the results were expressed as the fold change calculated with the 2−∆∆Ct method relative to a control sample. Meanwhile, *18S r-RNA* was used as an internal normalization control. All primers were purchased from Searchbio, Gyeonggi, Korea. Quantitative real-time PCR was performed according to the forward and reverse primer sequences listed in Supplementary Tables S1 and S2.

## *4.7. Evaluation of Autophagy by Immunocytochemistry (ICC), Flow Cytometry, and ELISA*

G-361 melanoma cells were used to monitor autophagy fluorescence (a cyto-ID®autophagy detection kit specifically measures autophagic vacuoles and autophagic flux). To get insight into the functional role of autophagy in the mentioned groups, we analyzed autophagy in human melanoma using the Cyto ID autophagy detection kit, which specifically tags the formation of autophagosomes (LC3B formation). This kit contains the anti-LC3B (anti LC-3II) antibody which stains autophagic sites and CTCF values were determined using image-J software. The melanoma cells were plated in 35 mm<sup>2</sup> Petri dishes and treated with CAP, SN, and CAP + SN for 24 h, respectively, as per the standard experimental procedures mentioned above. Rapamycin (500 nM) and chloroquine (10 µM) were added to positive control samples and acted as autophagy inducers. After 18 h with autophagy inducers and 24 h with CAP, SN, and CAP + SN, the media was replaced and washed with 1× assay buffer (in 5% FBS), which was further kept in 1 mL 1× assay buffer (without FBS). The cells were stained with Cyto-ID®green detection reagent and counterstained with Hoechst 33342. The autophagy was

determined by using an Olympus IX83-FP confocal microscope (Tokyo, Japan) and flow cytometry was performed by BD FACSVerse using the FACS suite software (Becton Dickinson and Co., Franklin Lakes, NJ, USA).
