Gradient polyacrylamide gel

Whole cell extracts were prepared from HMEC and MDAMB231 cells using SDS Lysis buffer supplemented with protease inhibitor (Sigma-Aldrich, St. Louis, MO). Equal amounts of protein from whole cell extracts were separated by running on gradient polyacrylamide gels (Bio-Rad, Hercules, CA), and transferred to Amersham Hybond polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK). These blots were first incubated with either a 1:2000 of anti-P63 (39739) (Active Motif, Carlsbad, CA) or anti-beta-tubulin (MAB3408) (Millipore Corp., Billerica, MA) for overnight, and followed by incubation with their corresponding secondary antibodies, anti rabbit-HRP-conjugated antibody (diluted 1:2500) (sc-2030) and anti mouse-HRP-conjugated antibody (diluted 1:4000) (sc-2031) (Santa Cruz, Dallas, Texas). Proteins were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) and ChemiDoc XRS + Imaging System with Image Lab (Bio-Rad, Hercules, CA).

Total proteins were extracted from kidney tissues with a lysis buffer and then loaded onto gradient polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA). Separated proteins were transferred from gels to nitrocellulose membranes. The membranes were probed with primary antibodies against NOX4 (Novus Biologicals, Littleton, CO, USA), MnSOD (Abcam, Cambridge, UK), RIPK1 (Cell Signaling, Danvers, MA, USA), RIPK3 (Cell Signaling, Danvers, MA, USA), p-MLKL (Abcam, Cambridge, UK), MLKL (Cell Signaling, Danvers, MA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling, Danvers, MA, USA), and then incubated with horseradish peroxidase-conjugated secondary antibodies. GAPDH was used as an internal control. The protein bands were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Waltham, MA, USA). The signal intensities of the bands were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Hetero-spheroids were harvested and lysed in 200 μl of Radio-immunoprecipitation assay (RIPA) Buffer, sonicated for 30s on ice with a probe sonicator. Extracted concentration was measured using the BCA Assay Reagent (Pierce) following manufacturer’s protocol for a 96-well format. Subsequently, 50 μg of protein from each sample was loaded onto 4–20% gradient polyacrylamide gels (Biorad), and separated electrophoretically, transferred to a PVDF membrane. Transferred membranes were blocked with 5% non-fat milk, and probed with β-catenin (R&D Biosystems) overnight at 4 °C, washed with TBST buffer, and probed with an appropriate HRP-conjugated secondary antibody. β-Actin was used as a loading control to determine changes in β-catenin expression among samples. ECL reagent (Pierce Protein Biology) was used to visualize bands in a Biorad ChemiDoc Touch instrument. Digital images acquired were processed through NIH Image J, and band analysis tools were used for densitometry. Band densities were normalized against the loading control β-Actin, to determine changes.

A biodegradable polyester urethane block copolymer, commercially available as DegraPol15 (DP) (M_w = 65 kDa) was kindly provided Ab Medica, Italy. The polymer was produced according to the procedure described earlier^{25 ,44} . Chloroform, ≥99.8% (0.5–1.0% ethanol as stabilized), 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, ≥95%, RPMI vitamins solution, RIPA buffer, phosphatase inhibitors and 0.5 M EDTA solution were purchased from Sigma-Aldrich, Switzerland. Recombinant human PDGF-BB and PDGF-BB ELISA kit were purchased from PeproTech. Ham’s F12 cell culture media, gentamicin, amphotericin B and Fetal Bovine Serum (FBS) were bought from Biowest, while non-essential amino acids solution, Pierce micro BCA protein assay and Pierce ECL Western Blotting Substrate were purchased from Thermo Scientific. Primaria^TM tissue culture plates were purchased from Corning. Primary antibodies used for western blot included rabbit anti Akt (9272 S, Cell Signaling Technology), rabbit anti pAkt (Ser473) (4060 S, Cell Signaling Technology) and rabbit anti GAPDH (G9545, Sigma Aldrich). Donkey anti rabbit antibody conjugated with HRP (Jackson) was used as secondary antibody for western blot. 4–20% gradient polyacrylamide gels were purchased from Bio-Rad, Switzerland.