Panc 1

Human pancreatic cancer cell lines, PANC1 and AsPC1 were obtained from Korea Cell Line Bank and maintained in RPMI1640 medium (WELGENE, Daegu, Korea) containing 10% heat-inactivated fetal bovine serum (Life Technologies, Grand Island, NY, USA) and antibiotics (100 U/ml of penicillin and 100 μg/ml streptomycin; Life Technologies). They were incubated at 37°C and 5% CO₂. AsPC1 cells were cultured, and stained with anti-CD133 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min on ice with rotation. Followed by washing with a buffer (phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.01% sodium azide) twice, CD133+ AsPC1 cells were isolated by FACSAria (BD Bioscience). Isolated CD133+ AsPC1 cells were incubated in CO₂ incubator, and subcultured for tumor xenograft.

Human PDAC cell lines (PANC-1, MIA PaCa-2, Capan-1, and Capan-2) and normal pancreatic ductal epithelial cell lines (H6c7) were purchased from the Korean Cell Line Bank (Seoul, South Korea) and Kerafast (Boston, MA, USA), respectively. PDAC cell lines were cultured in DMEM (PANC-1 and MIA PaCa-2) or RPMI-1640 medium (Capan-1 and Capan-2) supplemented with 10% FBS and 1% penicillin/streptomycin. H6c7 cells were maintained in keratinocyte serum-free medium supplemented with rEGF and BPE. All the cells were incubated at 37 °C under a 5% CO2. For measurement of cell viability, cells were seeded in 96-well plate in 1% FBS containing media. After cells were treated with vehicle or drugs for 48 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Merck, Burlington, MA, USA) dye solution was added. After 4 h, the solution was removed, and dimethyl sulfoxide was added to dissolve the formazan crystal. The absorbance was measured at 540 nm using a microplate reader (Versamax, Molecular Devices, Inc., San Jose, CA, USA).

Human pancreatic cancer cell lines (AsPC-1, Capan-1, Capan-2, Panc-1, SNU-213, SNU-324, and SNU-410) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). A human kidney cell line (293T) was obtained from the American Type Culture Collection (Manassas, VA). The cells were grown in DMEM (Panc-1 and 293T) or RPMI 1640 (AsPC-1, Capan-1, Capan-2, SNU-213, SNU-324, and SNU-410) medium supplemented with 10% fetal bovine serum (Gibco-BRL, Gaithersburg, MD, USA), 1 × 10⁵ unit/L penicillin, and 100 mg/L streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere containing 5% CO₂. Polyclonal antibodies for DUSP28 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and monoclonal antibodies for DUSP28 were purchased from Calbiochem (La Jolla, CA). Antibodies for caspase-3, cleaved caspase-3, phospho-ERK (Thr202/204), ERK, phospho-AKT (Ser473), AKT, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA). Doxorubicin and gemcitabine were from Sigma-Aldrich (St. Louis, MO, USA). U0126 and U0124 were purchased from Calbiochem (La Jolla, CA).

Cell culture and cytotoxicity was conducted according to our previous report [43 (link),44 (link)]. Three cancer cell lines (AGS, HT-29 and PANC-1) were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). Cells were grown in high-glucose DMEM supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL) and 10% FBS. Then, the cells were incubated at 37 °C (under 5% CO₂) and preserved at approximately 80% confluence prior to trypsin/EDTA treatment. Each cancer cell line was seeded on 96-well plates with a density of 5.0 × 10³ cells per well. Then, the incubation was performed for 24 h in a 37 °C incubator under CO₂ (5%). Five different concentrations of each nanoparticle solution (50.0, 25.0, 12.5, 6.25, and 3.12 μM Au or Ag) were treated on the cells [43 (link),44 (link)], and the treated cells were incubated in a 37 °C incubator for an additional 24 h under CO₂ (5%). The cells that were not treated were used as a control. Next, an MTT reagent (10 μL, 5% in deionized water) was added, and the incubation was conducted for an additional 3 h in a 37 °C incubator under CO₂ (5%). A multi-detection microplate reader was used to record the absorbance at 570 nm (Synergy HT, Bio Tek Instruments, Winooski, VT, USA). All cytotoxicity experiments were performed in triplicates.

Fetal bovine serum (FBS), antibiotics (penicillin/ streptomycin), and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Gibco BRL (Invitrogen Corp., Carlsbad, CA, USA). L929 cells (mouse connective tissue normal cell line, KCLB no.10001), A549 (human lung carcinoma cell line, KCLB no.10185), PANC-1 (human pancreas carcinoma cell lines, KCLB no.21469), and HCT116 (human colon carcinoma cell line, KCLB no.10247) were obtained from the Korean Cell Line Bank (KCLB). L929, PANC-1 cells were cultured in DMEM (Dulbecco Modified Eagle Medium) and A549, HCT116 cells were cultured in RPMI 1640 (Roswell Park memorial Institute 1640 Medium) supplemented with 10% FBS and 1% penicillin/ streptomycin. Cells were cultured at 37 °C in 5% CO₂ and changed fresh medium every 2 to 3 days. PuC and OPuC were dissolved in DMSO and diluted in serum-free (SF) medium until the DMSO concentration reached under 0.1%. All reported concentrations referred to free Ce6 equivalents. Untreated cells were kept in the dark and used as a reference standard.

The human pancreatic cancer cell line PANC-1 was obtained from the Korean Cell Line Bank (KCLB Cat# 21469) and grown in DMEM (Welgene, Daegu, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Welgene, Daegu, Korea) and 1% penicillin‒streptomycin solution (Gibco, Thermo Fisher, Waltham, MA, USA). Cells were cultured at 37 °C and 5% CO₂ in a humidified incubator.