Phosphate buffered saline (pbs)

Cell preparation tubes containing sodium citrate (Becton-Dickinson, Franklin Lakes, NJ, USA) were used to isolate PBMCs from whole blood. For storage, liquid nitrogen was used to freeze PBMCs in 10% RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA), 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA), and 80% fetal bovine serum (FBS) (Lonza, Cologne, Germany). PBMCs were cultured in RPMI 1640 medium containing 2 mM glutamine (Life Technologies), 50 μM 2-mercaptoethanol (Sigma-Aldrich), 50 mg/mL gentamicin sulfate (Lonza), and 10% FBS. After two washes in phosphate buffered saline (Welgene, Seoul, Korea), the PBMCs were resuspended at 1 × 10⁶ cells/mL in RPMI 1640 medium supplemented with 1% sodium pyruvate (Life Technologies), 1% MEM Nonessential Amino Acids Solution (Life Technologies), and 10% FBS, followed by incubation overnight. Assays for NK cells were always conducted following incubation for 16–20 h. To determine NK cell counts, peridinin chlorophyll protein-conjugated anti-CD3, and phycoerythrin-conjugated anti-CD56 and monoclonal antibodies (BD Biosciences, San Jose, CA, USA) were added to 200 mL of diluted blood that had been incubated on ice for 20 min, and the cells were then analyzed by flow cytometry (BD FACSCalibur, BD Biosciences). The NK cell abundance was calculated as (NK cells/mL sample) = [(CD3/CD56 cell count)/ bead count] × 100.

Immunohistochemistry was performed using the IHC Staining Kit (Dako, Santa Clara, CA, USA). Tissue sections were deparaffinized in xylene (Thermo), hydrated in phosphate‐buffered saline (Welgene), and blocked with Background Reducing Solution (Dako). The sections were incubated with anti‐CASQ2 (#NBP1‐87304; NOVUS), anti‐aSMA (#BS70000; Bioworld Technology), anti‐FSP1 (#BS7671; Bioworld Technology), anti‐HIF1α (#NB100‐131; NOVUS), anti‐vimentin (#5741; Cell Signaling Technology), anti‐pan‐cytokeratin (#M3515; Dako), and anti‐ki67 (#9027; Cell Signal Technology) at 4 °C overnight, followed by incubation with horseradish peroxidase‐conjugated anti‐secondary antibody (Dako). The signal was developed using diaminobenzidine and hydrogen peroxide, resulting in a brown precipitate. The sections were counterstained with hematoxylin (Dako), dehydrated, and mounted. The DAB area was quantified using IHC Toolbox in ImageJ software (NIH) with 20 random histological fields from five slides of tumor tissues per group [22 (link)].

Hydrocortisone was purchased from Sigma (St. Louis, MO, USA). HS was purchased from Life Technologies (Grand Island, NY, USA). Phosphate-buffered saline, 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Trypsin-EDTA), DMEM, antibiotic/antimycotic solution and FBS were purchased from WelGENE (Daegu, Korea).

The plasmids pT3/EF5a-TAZ^S89A, pT2/PI3KCA^E545K, and pPGK-SB13 were described previously [21 (link),22 (link)]. Hydrodynamic injection has also been previously described [21 (link)]. DNA mixtures of transposons (pT2- or pT3- plasmids) and transposase-encoding vector (pPGKSB13) were suspended in lactated Ringer’s solution and subsequently injected into the lateral tail veins of male 5–6-week-old mice (0.1 mL/g body weight). Mice were randomly assigned to hydrodynamic injection. Drugs were intraperitoneally administered daily beginning 5 weeks after hydrodynamic transfection. Doses of drugs administered was 50 mg/kg/day for vismodegib. All drugs were purchased from Selleckchem. All mice in the control group received an equal volume of 10% DMSO in phosphate-buffered saline (Welgene, Gyeongsan, Korea) by intraperitoneal injection according to the same treatment schedule.

The α-chitin powder from shrimp shells, sodium hydroxide (NaOH), dopamine hydrochloride, 2,3-dimethylmaleic anhydride, N, N′-dimethylformamide (DMF), trimethylamine (TEA), pyridine, anhydrous diethyl ether, gold (III) chloride hydrate (HAuCl₄), sodium citrate, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), sodium tetraborate, bovine serum albumin (BSA), 9,10-dimethylanthracene, formaldehyde, and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid (HCl) was bought from Daejung (Seoul, Korea). Ce6 was acquired from Frontier Scientific Inc. (Logan, UT, USA). The BCA protein assay kit was bought from Thermo Fisher Scientific Inc. (Walthan, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate-buffered saline (PBS), ethylenediaminetetraacetic acid (EDTA), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Welgene Inc. (Seoul, Korea). Wheat Germ Agglutinin-Alexa Fluor^® 488 conjugate (WGA-Alexa Fluor^® 488) was purchased from Life Technologies (Carlsbad, CA, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies Inc. (Santa Clara, CA, USA). FITC annexin V apoptosis detection kit I was acquired from BD Pharmingen (San Diego, CA, USA).