Image densitometer

Total protein extraction from cell cultures and immunoblots were performed according to standard methods, as described previously^{19 (link)}. Anti-rabbit and anti-mouse secondary antibodies were coupled to horseradish peroxidase (GE Healthcare, UK). Protein were visualized with an enzyme-linked chemiluminescence detection kit according to the manufacturer’s instructions (Amersham, UK). Chemiluminescence was monitored by exposure to films, and signals were analyzed under non-saturating condition with an image densitometer (Bio-Rad, USA).

Gel shift assays were carried out in a reaction mixture containing 10 µg of nuclear extract protein, 2 µL of 10× binding buffer (200 mM Tris–HCL pH 7.6, 500 mM KCL, 10 mM MgCl₂, 10 mM DTT, 2 mM EDTA, 0.1% (v/v), Triton X-100, 50% (v/v) Glycerol, and 5 mM spermidine), 2.5 µL poly dI·dC (1µg/L), and nuclease-free water in a total volume of 19 µL. After 10-min incubation at room temperature, 1 µL of ³²P-labeled oligonucleotides (40 fmols, approximately 50,000 cpm) was then added to the reaction and incubated at room temperature for 45 min before loading to the gel. The resulting DNA–protein complex was separated from free probe by electrophoresis on a 6% polyacrylamide gel with 0.5× TBE buffer. For binding competition analysis, non-radioactive oligonucleotides (50×–100× molar excess) were added as competitors to the binding reactions as indicated in the figure legends (Table 1). For antibody interaction studies, antibodies against C/EBP-α, C/EBP-β, C/EBP-γ, C/EBP-δ, and PPAR-α (Santa Cruz Biotechnology, Santa Cruz, CA 95060) were added into the reaction mixture during the pre-incubation time. The electrophoresis was performed at 4 °C, 240 V for 2.5 h. The gel was then dried and visualized following autoradiography for 16 h at − 80 °C without an intensify screen. The densitometry quantification was performed using a Bio-Rad Image Densitometer (Model GS-700).

For the cytotoxicity and genotoxicity assays, HTLA cells were treated for 24 hours with GEBR-32a (100 μM) at 37 °C. At the end of the incubation period, lactate-dehydrogenase release was measured in conditioned media using the Cytoxicity Detection Kit^PLUS (Roche, Germany) according to manufacturer protocols.
To evaluate genotoxicity, cells were processed for total protein extraction as described previously53 (link). Immunoblots were done according to standard methods, using the following antibodies: mouse monoclonal to gamma H2A.X (2F3, phospho S139) and rabbit polyclonal to Histone H2A.X (Abcam, UK); anti-mouse and anti-rabbit secondary antibodies coupled to horseradish peroxidase (GE Healthcare, UK). Proteins were visualized with an enzyme-linked chemiluminescence detection kit according to the manufacturer’s instructions (GE Healthcare). Chemiluminescence was monitored by exposure to films and signals were analyzed under non-saturating condition with an image densitometer (Bio-Rad, CA, USA).