Immunoblotting analysis of pathway activation in OSM-stimulated Edge cell cultures

OSM stimulation of Edge cell cultures
GCCs derived from glioblastoma edge samples were seeded at a density of 5 × 10⁵ cells per well in 6-cm culture dishes pre-coated with laminin. After a 24-hour incubation period, half of the wells were treated with 50 ng/mL (diluted in PBS) of human Oncostatin M (Peprotech, #300-10-50UG) and control cells were given PBS only (vehicle control (VC)). The treatment was refreshed every 48 hours for a total of 7 days. At the end of the treatment period, cells were harvested for subsequent analyses, including RNA and protein extraction, Extreme Limiting Dilution Assay (ELDA), invasion assays and cell viability assessment.

Western blot analysis
Edge cells from three independent OSM-stimulations have been analyzed. Cells were lysed using RIPA buffer (Thermo Scientific, 89900) supplemented with protease (Roche, CO-RO) and phosphatase inhibitors (Roche, PHOSS-RO). Protein concentrations were measured with the Pierce™ BCA Protein Assay Kit (Thermo Scientific, 23225). Equal amounts of protein samples were resolved on 4–12% Bis-Tris polyacrylamide gradient gels (NuPAGE, Thermo Scientific) and transferred onto nitrocellulose membranes (Novex, Thermo Scientific). Membranes were blocked with EveryBlot Blocking Buffer (BioRad, 12010020) and incubated overnight at 4°C with the following primary antibodies: rabbit anti-pSTAT3 (#9145, CST, 1:1000 dilution), mouse anti-STAT3 (#9139, CST, 1:1000 dilution), rabbit anti-pERK1/2 (#9101, CST, 1:1000 dilution), rabbit anti-ERK1/2 (#9102, CST, 1:1000 dilution), rabbit anti-pAKT(#4060, CST, 1:2000 dilution), rabbit anti-AKT(#4691, CST, 1:1000 dilution) and rabbit anti-GAPDH (#5174, CST, 1:1000 dilution). Following primary antibody incubation, membranes were incubated with HRP-conjugated secondary antibodies (anti-mouse, GE Healthcare, #NXA931, 1:5000 dilution; anti-rabbit, GE Healthcare, #NA9340, 1:5000 dilution) and developed using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific, 34095). Signals were visualized with the Amersham™ Imager 680 system (GE Healthcare). For membrane reprobing, Restore™ Western Blot Stripping Buffer (Thermo Scientific, 21059) was used according to the manufacturer’s protocol.

Raw, unedited immunoblotting images with annotated molecular weight markers were assembled in Adobe Illustrator. For each blot, an arrow denotes the band corresponding to the expected molecular weight of the target protein, as reported by the antibody manufacturer. The arrow-denoted band was selected for quantification using ImageJ2 software (Version 2.14.0). For the first membrane, pSTAT3, STAT3 and GAPDH were probed sequentially and stripped in this exact order. For the parallel membrane, pERK, ERK and GAPDH were probed and stripped in this exact order. For the final parallel membrane, pAKT, AKT and GAPDH were probed and stripped in this exact order.