4 hydroxynonenal

Both 8-hydroxy-2′–deoxyguanosine (purity ≥ 98%) and 5-hydroxymethyluracil (purity 97%) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 8-hydroxy-2′-deoxyguanosine-15N5 (purity 95%) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). 8-hydroxyguanosine (purity ≥ 98%), 8-isoprostane (purity ≥ 99%), and 8-isoprostane-d4 (purity ≥ 99%) were obtained from Cayman Chemical (Ann Arbor, MI, USA). 8-hydroxyguanosine-13C,15N2 (purity 98%), 4-hydroxynonenal (purity ≥ 98%), and 4-hydroxynonenal-d3 (purity 97%) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Methanol (LC-MS grade) was purchased from Burdick and Jackson (Muskegon, MI, USA). Formic acid and ammonium hydroxide were obtained from Sigma-Aldrich (St. Louis, MO, USA). Strata-X-A cartridges (33 µm Polymeric Strong Anion, 200 mg/3 mL; 8B-S123-FBJ), analytical columns (Kinetex phenyl-hexyl column, 100 mm × 4.6 mm, 2.6 µm particle size; 00D-4495-E0), and Kinetex C18 column, (100 mm × 4.6 mm, 2.6 μm particle size, 00D-4462-E0) were purchased from Phenomenex (Torrance, CA, USA).

Cells were seeded on 4 chamber slides (Bio Basic Inc., Markham, ON, Canada, Cat. No. SP41215) 24 hrs prior to experimentation. Cells were fixed with 3.7% formaldehyde at room temperature, followed by permeabilization with 0.15% Triton X-100 for 2 minutes, and then blocked with 5% bovine serum albumin (BSA) for 1 hr at room temperature. Cells were incubated for 1 hr at room temperature in the following primary antibodies: beclin-1 (mouse IgG, 1 : 500, Cat. No. sc-48342), C-Jun terminal kinase 1 (JNK1) (mouse IgG, 1 : 500, Cat. No. sc-137018), p21 (mouse IgG, 1 : 250, Cat. No. sc-817) (Santa Cruz Biotechnologies), 4-hydroxynonenal (rabbit IgG, 1 : 500, Cat. No. ab46545), and LC3B (rabbit IgG, 1 : 500, Cat No. ab192890) (Abcam Inc.). Cells were washed with PBS and incubated with horse anti-mouse FITC (1 : 500, MJS BioLynx Inc., Cat. No. Fl-2000) and/or a goat anti-rabbit Alexa Fluor™ 568 (1 : 500, Thermo Scientific Canada, Cat. No. A11011) secondary antibody for 1 hr at room temperature. Cells were washed again with PBS and incubated with 10 μM Hoechst 33342 (Molecular Probes, Eugene, OR, USA Cat. No. H3570). Cells were imaged using epifluorescence microscopy via a Leica DMI6000 B inverted microscope (Leica Microsystems, Concord, ON, Canada). Fluorescence was quantified in images captured using ImageJ software.