L 054 264

Cells were treated with 10 nM dermorphin [Y(d-ALA)FGYPKC; GenScript] or 10 nM (1R,1′S,3′R/1R,1′R,3′S)-L-054,264 (Tocris) individually and in combination for the indicated times. Cells were lysed for immunoblotting with phospho-ERK1/2, total ERK1/2, phospho-EGFR, and total EGFR. Blots were imaged on film and quantified using the Image Lab software. ERK1/2 and EGFR phosphorylation at each time point was quantified and normalized by calculating the ratio of pERK1/2 over total ERK1/2 and pEGFR over total EGFR in each lane. The data represent the percentage of maximum response to dermorphin treatment. The p values were obtained using the single-tail t test for the same time point between dermorphin activation and either L-054,264 or combined L-054,264 and dermorphin activation.

Nuclear and cytoplasmic extracts were made according to previously published protocols (Smith et al., 2004 (link); Rozenfeld and Devi, 2007 (link)). Briefly, PANC-1 cells were treated with 10 nM dermorphin or 10 nM (1R,1′S,3′R/1R,1′R,3′S)-L-054,264 (Tocris, Bristol, UK) individually and in combination for the indicated times. After the treatment, cells were washed in ice-cold PBS three times and scraped into lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 1 mM ethylene glycol tetraacetic acid, 1 mM sodium orthovanadate, and a protease phosphatase inhibitor tablet [ThermoFisher Pierce]) and incubated on ice for 10 min. The lysate was then homogenized and centrifuged at 375 × g for 5 min. The pellet consisting of the nuclear fraction was washed five times with lysis buffer containing 0.1% NP-40 to remove any nonnuclear contamination and resuspended in lysis buffer containing NP-40. The soluble fraction was centrifuged twice at 375 × g to remove nuclear contamination and used as the cytosolic fraction. Fractions were then used for immunoblotting for anti–phospho-ERK1/2, anti–phospho-p90RSK (Thr-573; rabbit polyclonal; Cell Signaling, Danvers, MA), and anti–β-actin as a loading control for the cytoplasmic fraction, and Histone H3 antibody (rabbit monoclonal; Cell Signaling) was used as a loading control for the nuclear fraction.

PANC-1 cells were treated with 1) 10 nM dermorphin (Y(D-ALA)FGYPKC; GenScript, Piscataway, NJ) and 2) 10 nM (1R,1′S,3′R/1R,1′R,3′S)-L-054,264 (Tocris) individually and in combination for 24 h. Cells were lysed as described for immunoblotting with EMT marker antibodies (vimentin rabbit monoclonal, E-cadherin rabbit monoclonal, N-cadherin rabbit monoclonal; Epithelial-Mesenchymal Transition Antibody Sample Kit; Cell Signaling) according to the manufacturer’s protocol. Blots were imaged using the Image Lab software (Bio-Rad).