4.8. Western Blotting

Whole cell lysates from S2VP10, S2CP9, MiaPaCa-2, Panc-1, and ES-2 (positive control IGF1-R) and MCF-7 (negative control IGF1-R) cells were collected using a cell scraper as in [59] to maintain receptor integrity. Whole cell lysates were used to evaluate IGF1-R, HIF-1α, and BNIP3 expression with Western blot. As both chronic expression of HIF1-α and low/lack of BNIP3 expression have been associated with poor prognosis, chemoresistance, and using autophagy as a survival mechanism in pancreatic cancer [37,44,60], S2VP10, S2CP9, MiaPaCa-2, and MCF-7 (positive control BNIP3 and responsive to hypoxia) were cultured at 20%, 10%, and 1% O2 for 5 h at 37 °C to evaluate HIF1-α and BNIP3. Approximately 100 µg of total protein was dissolved in deionized water, loading buffer, and reducing agent (Life Technologies, Grand Island, NY, USA). Duplicate Western blot gels were assessed using 50 µg/protein/lane. Proteins were separated using NuPage 4–12% Bis–Tris gel and transferred onto a nitrocellulose membrane by iBlot (Life Technologies). The membranes were blocked in blocking buffer (Li-Cor) for 30 min and incubated overnight at 4 °C with primary antibodies. Primary antibodies utilized were IGF1-R (ab182408, Abcam), HIF1-a (NB100-479, Novus), and BNIP3 (NB100-56150, Novus). The membrane was washed 3× with Tris-buffered saline (20 mM Tris–HCl, 150 mM NaCl in diH2O) for 30 min, incubated with appropriate secondary antibody for 1 h, washed 3× with TBS for 30 min, and scanned using Li-Cor Odyssey infrared scanner. Dosimetry was performed using Li-Cor software. Western blots were conducted three independent times.

To evaluate the method of cell death following treatment with echinomycin, SDC1-Lip with echinomycin, standard of care Gemzar, or bafilomycin a1, S2VP10, S2CP9, and MiaPaCa-2 cells were assessed in triplicate using Western blot. The cells were plated at a density of 5 × 10⁵ cells per well in a 6-well plate 24 h before protein harvest. Cells were treated with 300 nM Gemzar, 5 nM bafilomycin a 1, 1 nM echinomycin, or SDC1-Lip containing 1nM echinomycin under normoxic conditions of 5% CO₂ (80% N₂ and 20% O₂) for 24h. Protein was harvested, and Western blotting was conducted as above in duplicate. Antibodies utilized were: LC3-I/II (NB100-2220, Novus), p62 (NBP1-49956, Novus), ATG 7 (MAB6608, Novus), ATG 12 (NBP2-15501, Novus), caspase-3 (ab184787, Abcam), cleaved caspase-3 (9661, Cell Signaling Technologies), caspase-9 (B100-56119, Novus), and B-actin (PA1-183, Invitrogen).