Protease and phosphatase inhibitors cocktail

HIF-1α protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions^{49 (link)}. In order to avoid further HIF-1α degradation during whole cell lysates preparation, cells were lysed directly with NuPAGE® LDS Sample Buffer (1X) (Thermo Fisher Scientific, San Jose, CA, USA) in the presence of protease and phosphatase inhibitors cocktail (Cell Signaling Technology, Danvers, MA, USA), similarly to ref. 50 (link). Protein extracts were separated by electrophoresis on 4–12% NuPAGE Bis-Tris Mini Gel and then transferred onto nitrocellulose membranes using iBlot transfer stacks (Life Technologies, Gaithersburg, MD, USA). Membranes were probed with various primary antibodies overnight at 4 °C. All washes and secondary antibody incubations were performed using the SNAP i.d. quick immunoblot vacuum system (Millipore, Billerica, MA, USA). Bands were developed using Clarity Western ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA). Visualization and quantitative densitometry analysis were performed with Molecular Imager® Gel Doc™ XR System v5.2.1 (Bio-Rad Laboratories, Hercules, CA, USA).

Total protein extraction was performed by homogenizing cells lysis buffer containing 1× protease and phosphatase inhibitors cocktail (lysis buffer 10×; Cell Signaling Technology, Danvers, MA, USA). After sonication, the homogenates were centrifuged at 300 g for 5 min at 4 °C. Protein concentrations were determined using BCA (bicinchoninic acid assay) to measure the concentration of protein in a solution. Lysates obtained from 2 subpopulations were analyzed in denaturing condition by SDS-PAGE and transferred on to nitrocellulose membranes (Amersham Bioscience, Little Chalfont, UK). For the experiments with CD44v8-10-knocked-down PC3 cells, the total lysates were prepared 72 h after siRNA/scramble transfection. Membranes were incubated with primary antibodies rabbit anti-human-phospho-β catenin (Ser 33/37 Thr 41) and rabbit anti-human total β catenin, rabbit anti-human N-cadherin, rabbit anti-human P-PARγ, and rabbit anti-human RUNX2 (all from Cell Signaling Technology), and rabbit anti-human MMP-13 (Abcam, Cambridge, UK) followed by goat anti-Rabbit IgG H + L HRP-conjugated secondary antibody (Bio-Rad, Hercules, CA, USA) and visualized by ECL (Western nova 2.0; Cyanagen, Bologna, Italy) using Chemidoc Gel Imaging System Image Lab version 5.2.1. software (Bio-Rad). Densitometric analysis of immunoblots was performed by ImageJ.

A number of 4.8 × 10⁵ (MIA PaCa-2) or 6 × 10⁵ (PANC-1) cells were plated in 100 mm plates and left free to attach for 24 h. In the next day, media was replaced with a fresh one containing AdipoR, Gem, and the combination in doses and timelines reported in the Results section, and Figure legends. At every experimental point, cells were collected and spun down at 1,500 RPM for 5 min. Pellets were later resuspended in 3–5 volumes of RIPA buffer (R0278; Sigma-Aldrich) supplemented with protease and phosphatase inhibitors cocktail (#5872; Cell Signaling Technology). After 30 min, samples were further centrifuged at 14,000 RPM for 15 min at 4°C, and the supernatant was recovered and assessed for the relative protein content by Bradford Assay (39222; SERVA). Protein samples were first mixed 1:1 with Laemmli 2× (S3401; Sigma-Aldrich) and later boiled at 95°C for 6 min.