100 mm plates

VCaP or VCaP/GanR were plated at 5×10⁶ in 100 mm plates (BD Falcon) in media containing 10% FBS. The following day medium was replaced with DMEM containing 10% FBS and 500 nmol/L ganitumab or control IgG1 antibody and incubated for six hours prior to harvest. Proteome Profiler Human Phospho-Kinase Arrays ARY003B were obtained from R&D systems and performed following the manufacturer's protocol. Densitometry was performed using Adobe Photoshop CS3.

The cellular apoptosis was evaluated following the protocol as previously described by our group (Rosenberger et al., 2019 (link)). Briefly, 6,250 cells/cm² were seeded in 100 mm plates (Falcon, Franklin Lakes, NJ, United States, Cat. #353003) in maintenance medium. After 48 h, the culture medium was removed, and the cells were washed three times with phosphate-buffered saline (PBS 1×) before starting the culture in the different induction medium: (a) DMEM high glucose + 1% L-glutamine; (b) Oxium^TMEXO (Consorcio Regenero S.A., Las Condes, Santiago, Chile; patent No. PCT/CL2019/100175, Tapia-Limonchi et al., 2019 ); or (c) commercial medium (RoosterBio Inc., Frederick, MD, United States, Cat. #M2001). After 6 days, cell supernatants were mixed with the trypsinized cells in order to include detached dead cells in the analysis. Then, cells were stained with Annexin V-APC (BioLegend, San Diego, CA, United States, Cat. #640920) and 7-aminoactinomycin D (7-AAD) (BioLegend, San Diego, CA, United States, Cat. #420403) in Annexin V binding buffer (BioLegend, San Diego, CA, United States, Cat. #422201). The analysis was performed by flow cytometry using a FACSCanto^TM II cytometer (BD Biosciences, San Jose, CA, United States). The data acquired were analyzed using the FlowJo software V10 (Tree Star, Ashland, OR, United States).