*4.9. Mitochondrial Respiratory Complex Activity Assay*

Mitochondrial complexes activities were determined in mitochondrial membrane-enriched fractions from PC12 cells [45]. Aliquots of trypsinized cells were washed with ice-cold PBS, frozen in liquid nitrogen, and kept at −80 ◦C until use. To isolate the mitochondrial membrane-enriched fractions, cell pellets were thawed at 2–4 ◦C, suspended in 1 mL of 10 mM Tris–HCl (pH 7.5), supplemented with 1 mg/mL BSA, and exposed to ultrasound energy for 15 s at 0 ◦C. The ultrasound-treated cells were centrifuged (10 min at 600× *g*, 4 ◦C). The supernatant was obtained and centrifuged again (10 min at 14,000× *g*, 4 ◦C) to collect a mitochondrial pellet that was suspended in 0.1 mL of the respiratory medium. The activity of NADH:ubiquinone oxidoreductase (complex I) was measured in 40 mM potassium phosphate buffer, pH 7.4, 5 mM MgCl2, in the presence of 3 mM KCN, 1 μg/mL antimycin, 200 μM decylubiquinone, using 50 μg of mitoplast proteins, by following the oxidation of 100 μM NADH at 340–425 nm (Δε = 6.81 mM−<sup>1</sup> cm−1). The activity was corrected for the residual activity measured in the presence of 1 μg/mL rotenone. [46]. Succinate-cytochrome c oxidoreductase (complex II + III) activity was determined by following its reduction at 550–540 nm in the presence of cytochrome c (Δε = 19.1 mM−<sup>1</sup> cm<sup>−</sup>1). The activity was also determined in the presence of antimycin A. The activity of Cytochrome c oxidase (complex IV) was established following the ferrocytochrome c oxidation at 550–540 nm (Δε = 19.1 mM−<sup>1</sup> cm<sup>−</sup>1). Complex V activity (ATP hydrolase activity) was measured by an ATP-regenerating system. Frozen and thawed cells were suspended (at 0.1 mg protein/mL) in a buffer consisting of 375 mM sucrose, 75 mM KCl, 30 mM Tris–HCl pH 7.4, 3 mM MgCl2, 2 mM PEP, 55 U/mL lactate dehydrogenase, 40 U/mL pyruvate kinase, 0.3 mM NADH. The reaction was started by the addition of 1 mM ATP and the oxidation of NADH was followed at 340 nm [45].

#### *4.10. Protein Extraction and Western Blot Analysis*

After treatment with DEHP (85 μM), alone or combined to HE (1 μM), for 24 h at 37 ◦C, PC12 cells (1 <sup>×</sup> 105) in 6-well plates were harvested, washed with PBS, and lysed in 100 <sup>μ</sup>L lysis buffer (Hepes 0.5 M containing 0.5% Nonidet-P40, 1 mM PMSF, 1 mg/mL aprotinin, 2 mg/mL leupeptin, pH 7.4), and incubated 20 min in ice before centrifugation. Protein concentrations were determined in cell lysates using Protein BioRad assay. Western blot was carried out as described in [39], proteins extracted for each sample, at equal concentration (50 μg) were boiled for 3 min in sample buffer (containing 40 mM Tris–HCl pH7.4, 2.5 % SDS, 5 % 2-mercaptoethanol, 5 % glycerol, 0.025 mg/mL of bromophenol blue), and then separated on a polyacrylamide mini gels precasting 4-20 % (codNB10420 NuSept Ltd. Australia). Separated proteins were transferred onto nitrocellulose membrane (BIO-RAD, Hercules, CA, USA) in transfer buffer containing (0.05 % SDS, 25 mM Tris, 192 mM glycine, and 20 % v/v methanol). The transfer of the proteins on the nitrocellulose membrane was confirmed by staining with Ponceau Red which was then removed by three washes in PBS (phosphate buffered saline) for 5 min each. Membranes were then incubated for 1 h at room temperature in 20 mM Tris pH 7.4, 150 mM NaCl, and Tween 20 (TBS-T) containing 2 % milk powder and incubated with appropriate primary anti-Hsp70 (SC-10789, Santa Cruz Biotech. Inc), anti-heme oxygenase-1 (HO-1) (SC-10789, Santa Cruz Biotech. Inc), anti-Thioredoxin (Trx) (Sc-13526, Santa Cruz Biotech. Inc.), anti-Sirt1 (SC-74465, Santa Cruz Biotech. Inc.), anti-Bax (SC-7480, Santa Cruz Biotech. Inc, anti-Bcl-2 (SC-7382, Santa Cruz Biotech. Inc), anti-p53 (SC-126, Santa Cruz Biotech. Inc), and anti-caspase-3 polyclonal (Santa Cruz Biotech. Inc.) overnight at 4 ◦C. The same membrane was incubated with a goat polyclonal antibody anti-beta-actin (SC-1615 Santa Cruz Biotech. Inc., Santa Cruz, CA, USA) to verify that the concentration of protein loaded in the gel was the same in each sample. Excess unbound antibodies were removed by three washes are with TBS-T for 5 min. After incubation with primary antibody, the membranes were washed three times for 5 min. in TBS-T and then incubated for 1 h at room temperature with the secondary polyclonal antibody conjugated with horseradish peroxidase (dilution1:500). The membranes were then washed three times with TBS-T for 5 min. Finally, the membranes were incubated for 3 min with Super Signal chemiluminescence detection system kit (Cod34080 Pierce Chemical Co, Rockford, IL, USA) to display the specific protein bands for each antibody. The immunoreactive bands were quantified by capturing the luminescence signal emitted from the membranes with the Gel Logic 2200 PRO (Bioscience) and analyzed with Molecular Imaging software for the complete analysis of regions of interest for measuring expression ratios.
