Protein Oxidation and Autophagy Regulation in CML Cells

Lysate from CML cells, treated or not treated with OLDA at the IC₅₀ dose, was extracted using lysis buffer (10 mM Tris; 100 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM NaF; 20 mM Na₄P₂O₇; 2 mM Na₃VO₄; 1% Triton X-100; 10% glycerol; 0.1% SDS; 0.5% deoxycholate; and 1 mM PMSF) containing protease-inhibitor cocktail (Euroclone). Proteins were separated on 8%–14% SDS-polyacrylamide gels and transferred using Bio-Rad systems. Non-specific binding sites were blocked with 5% low-fat dry milk or 5% BSA in PBS containing 0.1% Tween 20 for 1 h at room temperature. Membranes were incubated overnight at 4°C with anti-LC3, anti-ATG12, anti-ATF4, anti-BiP, anti-γH2AX, anti-caspase 3, anti-phospho-ubiquitin (pSer65), or anti-GAPDH Abs, followed by corresponding HRP-conjugated secondary Abs. In some experiments, Western blot analysis was performed on siTRPV1 and siGLO (control) CML cells treated with OLDA or vehicle to assess BiP expression levels. Moreover, lysates from CML cells were treated with OLDA at the IC₅₀ dose and imatinib mesylate (0.5 µM), either alone or in combination, for 24 h were incubated with anti-γH2AX and anti-caspase 3.
In addition, the protein oxidation products were identified in CML cells, which were treated as described previously, by scanning carbonyl groups using the OxyBlot^TM Protein Oxidation Detection Kit according to the manufacturer’s instructions. In brief, dinitrophenylhydrazine was added to the crude total proteins (20 μg) to derive the carbonyl groups from the protein side chains. Carbonylated proteins were resolved by SDS-polyacrylamide gel electrophoresis, and Western blot analysis was performed using the provided anti-DNP antibody (1:150).
The detection was performed using LiteAblot PLUS kit, ChemiDoc, and Quantity One software (Bio-Rad). GAPDH was used as the loading control. SHARPMASS VI–VII (Euroclone) and SeeBlue Plus2 (Invitrogen) were used as pre-stained protein markers.