Mia paca 2 cell line

Pancreatic adenocarcinoma (MIA PaCa-2) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM, supplemented with 100 units of penicillin, 100 µg/mL of streptomycin, 300 µg of L-glutamine, 10% heat-inactivated FBS (ATCC) and 2.5% horse serum. Exosomes were isolated from YUSAC 2, a melanoma cell line obtained from Dr. Doug Grossman at the Huntsman Cancer Institute in Salt Lake City, Utah. The two cell line derivatives from YUSAC 2 were designed to overexpress either Survivin-WT (4C7 cells) or Survivin-T34A (F5C4 cells) in the absence of tetracycline (tet), otherwise only normal endogenous levels of Survivin are produced. YUSAC 2 cells were maintained in DMEM (CellGro, Manassas, VA) supplemented with 100 units of penicillin, 100 µg/mL of streptomycin, 300 µg of L-glutamine, 5% newborn calf serum (Thermo Scientific HyClone, Rockford, IL), 0.5 µg/mL tetracycline (tet-off system), 1.5 mg/mL Geneticin G418 (Teknova, Holister, CA) and 2 mM NaOH. YUSAC 2 cells were grown to 60% confluency in the presence of tetracycline in order to establish a healthy monolayer culture. After which, cells were washed carefully twice in PBS followed by the addition of media in the absence of tetracycline. All cells were grown at 37°C in a humidified atmosphere containing 5% CO₂.

MIA PaCa-2 cell line was purchased from the American Type Culture Collection (Manassas, VA), and MIA PaCa-2–resistant (MIA PaCa-2R) cells were generated from MIA PaCa-2 by incubating with GEM in a high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C, 5% CO₂, and 100% humidity and were split when confluent. HPDE cells were cultured in Keratinocyte-SFM–supplemented bovine pituitary extract and human recombinant EGF in an identical atmosphere. hPSCs were cultured in stellate cell basal medium supplemented with 10% FBS, stellate cell growth supplement, and 1% penicillin/streptomycin. A hypoxic chamber filled with 94% nitrogen, 5% CO₂, 1% O₂, and 100% humidity was used to generate hypoxic conditions as needed.
hPSC-conditioned medium was obtained using the following steps: Subconfluent PSCs were washed with PBS and incubated with stellate cell basal medium under above conditions for 48 hours. Then, the medium was collected and centrifuged for further use.

The pancreatic cancer MIA PaCa-2 cell line was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). The siRNA-Lfng and Notch3 intracellular domain (N3IC)-overexpressing MIA PaCa-2 cell models were established as previously described (3 (link)). The cells were cultured according to ATCC protocols in Dulbecco's modified Eagle's medium (DMEM) (Corning Incorporated, Corning, NY, USA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Inc., Flowery Branch, GA, USA) at 37°C with 100 U/ml penicillin and 100 µg/ml streptomycin maintained in a humidified environment containing 5% CO₂. For cell counting, MIA PaCa-2 cells were respectively treated with 100, 50, 25, 12, 6, 3, 1.5, 0.8, 0.4, 0.2 and 0.1 µM PI3K inhibitor AS-605240 (catalog no. S1410; Selleck Chemicals, Houston, TX, USA) at 37°C for 72 h. For wound healing assays, MIA PaCa-2 cells were treated with AS-605240 at a final concentration of 5 µM for 48 h.