4 hne

Culture media were precipitated with trichloroacetic acid (1 : 100, vol/vol, overnight incubation at −20°C). The precipitates were rinsed with acetone, prior to be resuspended into lysis buffer. Proteins extracted from cells or heart tissues (40 μg protein for each sample) were electrophoresed on a SDS-PAGE and transferred onto a PVDF membranes (Bio-Rad) and incubated with primary antibodies against phosphorylated PKCε (1 : 500, Santa Cruz, CA, USA), PKCε, cleaved caspase-3, β-actin (1 : 1000, all from Cell Signaling Technology, Danvers, MA, USA), or 4-HNE (1 : 500) (both from Abcam, Cambridge, MA, USA). Then, membranes were incubated with HRP-conjugated secondary antibodies and exposed with the Chemiluminescence Detection Kit (Millipore).

Lungs from each group of mice were homogenized in 500 μl lysis buffer (10 mM Tris-HCL pH 7.5, 120 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 1% Triton X-100) containing protease and phosphatase inhibitor cocktails (Roche) using a sonicator on ice. Samples were centrifuged at 14,000 g for 20 min at 4 °C, and the supernatants were collected. Sample protein concentrations were measured using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL), and 65-μg protein samples were separated by electrophoresis on 4–12% gradient Bis-Tris gels and transferred to nitrocellulose membranes. After blocking with 0.1% casein, membranes were incubated with primary antibodies against BiP, pIRE1α, CHOP, cleaved caspase-12 (Cell signaling Technology, Danvers, MA), sXBP1, NF-κ p65 (Santa Cruz Biotechnology, Santa Cruz, CA), 4-HNE (Abcam, Cambridge, MA), or β-actin (Sigma-Aldrich, St. Louis, MO). 4-HNE is stable, making its quantification more reliable than direct quantification of ROS
After washing, membranes were incubated with the appropriate fluorescent-conjugated secondary antibodies. Bands were detected using an Odyssey FC Imaging system (LI-COR, Lincoln, NE). Band intensities were quantified with densitometry and represented as fold changes relative to controls. The corrected band intensity data from two Western blots was used to plot each histogram.

Frozen kidney samples were cut into 7 µm sections and fixed with acetone at −20 °C for 10 min and blocked with 2% bovine serum albumin (BSA) in PBS (1 h, room temperature). Tissue was then incubated with primary antibodies against 4-hydroxynonenal (4-HNE, Abcam, Cambridge, UK) in 2% BSA in PBS for 1 h at room temperature. Secondary fluorescent-conjugated anti-rabbit Alexa Fluor^®647 (1:400; Invitrogen, Carlsbad, CA) was used for 40 min at room temperature in dark, followed by washing in PBS and distilled water and counterstaining with 4′,6-diamidino-2 phenylindole (DAPI; Invitrogen, Carlsbad, CA) for 5 min. Sections were then washed in PBS and distilled water before mounting with fluorescence mounting medium (Dako, Glostrup, Denmark). Images were obtained using Olympus FV1000 Confocal microscope at ×40 magnification (Olympus Corp., Tokyo, Japan).

Cells were seeded on 8-chamber slides (Bio Basic Canada Inc., Cat. No. SP41219, Markham, ON, Canada) 24 h prior to treatment. Following treatments of 24 or 48 h, cells were fixed with 3.7% formaldehyde prepared in 1X PBS for 15 min at room temperature, permeabilized with 0.15% Triton X-100 for 2 min and blocked with 5% bovine serum albumin for 1 h. Cells were washed with 1X Tris-Buffered Saline, 0.1% Tween 20 Detergent (TBST) and incubated for 1 h at room temperature with the following primary antibodies: LAMP1 (mouse IgG, 1:250, Cat. No. ab25630) (Abcam Inc., Cambridge, UK), LC3B (rabbit IgG, 1:500, Cat. No. ab192890) (Abcam Inc.), 4-HNE (rabbit IgG, 1:200, Cat. No. ab46545) (Abcam Inc.). Cells were washed with TBST and incubated with goat anti-rabbit Alexa Fluor™ 568 (1:500, Thermo Scientific Canada, Cat. No. A11011) and/or horse anti-mouse fluorescein isothiocyanate (1:500, MJS BioLynx Inc., Brockville, Canada, Cat. No. Fl-2000) secondary antibodies for 1 h at room temperature. Cells were washed with TBST and incubated for 2 min with 10 µM Hoechst 3342 (Molecular Probes, Cat. No. H3570, Eugene, OR, USA). Cells were washed with 1X PBS and imaged via epifluorescence microscopy with a Leica DMI6000 B inverted microscope (Leica Microsystems, Concord, ON, Canada). ImageJ software was utilized for fluorescent quantification.

The production of intracellular ROS (iROS) was measured by the cell-permeable fluorescence probe chloromethyl-29, 79-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Cat# C6827; Invitrogen) or 2′,7′-Dichlorofluorescin Diacetate (H₂DCFDA; Cat# 287810; Calbiochem) as described [27 (link)]. Briefly, mixed-glia cultures in black 96-well plates were pretreated with vehicle or G6PD inhibitors for 30 min followed by treatment with LPS for 6 h in phenol red-free medium. After incubated with 10 μM H₂DCFDA for 1 h at 37 °C, the fluorescence density was read at 488 nm for excitation and 525 nm for emission using a SpectraMax Gemini XS fluorescence microplate reader (Molecular Devices). Alternatively, microglia-enriched cultures grown in glass chambers were transfected with G6PD siRNAs or scramble siRNA for 30 h or pretreated with vehicle/G6PD inhibitors for 30 min followed by treatment for 18 or 24 h with LPS as described in the figure legend. The cultures were stained with 10 μM CM-H₂DCFDA or antibody against 4-hydroxynonenal (4-HNE; 1:500; Abcam). Brain sections were immune-stained for 3-nitrotyrosine (3-NT; 1:500; Abcam). Fluorescent images were collected using Zeiss LSM 780 or 880 confocal microscope and analyzed by using ImageJ software.

After deparaffinization and rehydration, the sections were boiled in citrate sodium buffer for 15 min for antigen recovery, and were immersed in 3% H₂O₂ for 10 min to quench endogenous peroxidase. Sections were then blocked with 10% serum at 37 °C for 1 h. The primary antibodies (Ki67, CST, Danvers, MA, USA, 1 :  200 dilution; RAD51, Abcam, 1 : 200 dilution; 4-HNE, Abcam, 1 : 10 dilution; Cleaved Caspase-3, CST, 1 : 300 dilution; γ-H2AX, Upstate Biotechnology, 1 : 200 dilution) were added to the sections and incubated overnight at 4 °C. After washing, the sections (Ki67, RAD51 and 4-HNE) were coated with a HRP-conjugated second antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1 : 200 dilution) and then incubated at 37 °C for 1 h. DAB was used to visualize immunoreactions. Sections (Cleaved Caspase-3, γ-H2AX) were then washed three times in PBS before incubating in the dark with a Rhodamine-labeled secondary antibody at a dilution of 1 : 300 in 5% bovine serum albumin in PBS for 60 min. The secondary antibody solution was then aspirated and the sections were washed four times in PBS. The sections were incubated in the dark with DAPI (1 mg/ml) in PBS for 5 min and coverslips were mounted with an antifade solution (Molecular Probes, Life Technologies, Carlsbad, CA, USA). Negative controls were performed by omitting the primary antibodies.

The abdominal fat mass (100 mg) of ICR mice was homogenized in 400 µl lysis buffer containing 4 µl protease inhibitor and 4 µl 0.5 M EDTA (ThermoFisher Scientific). Homogenates were subjected to 10% sodium dodecyl sulfate polyAcrylamide gel electrophoresis (SDS‐PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane (Sigma) and blocked with 5% (w/v) non‐fat milk before incubation overnight with primary antibodies to 4-HNE (1:2,000, Abcam, Cambridge, MA, USA) or β‐actin (1:1,000, Cell Signaling, Danvers, MA, USA). This was followed by treatment for 1 h with horseradish peroxidase (HRP)‐conjugated secondary antibody (goat anti‐mouse (1:5000). Protein bands were detected with a chemiluminescent detection reagent and Omega Lum C Imaging System (Gel Company, San Francisco, CA, USA). Densitometric analysis of the protein bands was performed using Image J software (National Institute of Health (NIH), Bethesda, MD, USA).