X ray film

To confirm cancer development in the tibia, rats were radiographed after 12 following implantation. After the rats were anesthetized with pentobarbital sodium, the right hind limbs were placed on X-ray film (Kodak, Italy) and exposed to X-ray for 1/20 s at 40 kVp. The images were taken from sham rats and BCP rats.

Genomic DNA was extracted from non-transformed and putative transgenic plants as
described by Dellaporta et al. [52 (link)].
Each genomic DNA sample (20 µg) was first digested with appropriate restriction
enzymes, electrophoresed on a 0.7% (w/v) agarose gel,
and transferred to a Hybond-N membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Membranes were soaked overnight in hybridization solution (6× SSPE, 5×
Denhardt’s Reagent, 0.5% SDS, 250 μg·mL⁻¹ salmon sperm
DNA, and 10% dextran sulfate) at 65 °C. DNA samples on the membranes were hybridized
with probe labeled with ³²P-dCTP using random-priming kit (Promega, Fitchburg,
WI, USA). Then the membranes were washed twice for 15 min with Buffer I (2 × SSPE and
0.1% SDS) at room temperature, and twice for 15 min with Bbuffer II (1 × SSPE and
0.1% SDS) at 65 °C. Visual results were displayed by X-ray film (Kodak, USA)
exposure.

SDS-PAGE was used to separate the proteins (20 μg) from each sample. The proteins were then transferred to polyvinylidene fluoride membranes, which were incubated with appropriate primary antibodies and an HRP-conjugated anti-mouse IgG secondary antibody. Immunoreactive bands were visualized with an enhanced chemiluminescence system (Biouniquer, China) and exposed to X-ray film (Kodak). Signal intensities were quantified by using the ImageJ program and normalized to the corresponding β-actin signal.

2x10⁵ YUMM3.3 cells were seeded in 24-well plates and incubated with 20 ng/ml IFN-γ (485-MI, R&D Systems), 10 μM SB-431542, 50 ng/ml of Activin-A for 24 h where indicated. Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (Roche, Basel, CH) and phosphatase inhibitors (Sigma-Aldrich). Proteins were separated on 9-12% SDS-PAGE gels under reducing conditions and transferred on nitrocellulose membranes. Membranes were blocked with 5% skim milk (Sigma) in Tris-buffered saline containing 0.1% Tween-20, before incubation with primary antibodies against γ-tubulin (Sigma GTU88) or pSTAT (9167S), STAT1 (9172), or STING (13647S) antibodies (all from Cell Signaling) for 2 hrs at room temperature or overnight at 4°C. Chemiluminescence was revealed on X-ray film (Kodak, Rochester, NY, USA) or ChemiDoc MP (Biorad, Hercules, CA, USA) using HRP-coupled secondary antibodies and ECL reagents (Thermo Fisher).

The Ad‐directed ING4 and OSM transgene expression in Hep‐2 cells was analysed by RT‐PCR and Western blot. For the RT‐PCR, the total cellular RNA was extracted using TRIzol, and the first‐strand cDNA was reverse transcribed with RNA as a template and Oligo d(T)₁₈ as a primer. The PCR amplification was carried out using cDNA as the template and primers specific for ING4 and OSM, respectively. For the Western blot, proteins were isolated from infected and uninfected Hep‐2 cells (1–2 × 10⁶), resolved by 12% sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% (w/v) nonfat dry milk in tris‐buffered saline containing 0.05% Tween 20 (TBST), the membrane was probed with primary antibody polyclonal mouse anti‐ING4 (1:1000) or anti‐OSM (1:1000) in blocking solution for 2 h, followed by incubation with horseradish peroxidase (HRP)‐conjugated secondary antibody (rabbit anti‐mouse) for another 2 h. The membrane was developed using a SuperEnhanced chemiluminescence detection kit. The immunoreactive bands were visualized after exposure of the membranes to Kodak X‐ray film.