Ab32516

Liver tissue was homogenized in HEPES buffer and proteins were separated by SDS-PAGE and transferred to PVDF membranes using the iBlot Dry Blotting System (Invitrogen AB, Lidingö, Sweden). Membranes were immunoblotted using the following antibodies: HMGB1 (ab79823, Abcam, Cambridge, UK), caspase-3 (#9662, Cell Signaling Technology, MA, US), apoptosis-inducing factor (AIF) (ab32516, Abcam, Cambridge, UK), Beclin-1 (ab207612, Abcam, Cambridge, UK) and B-cell lymphoma 2 (Bcl-2) (ab136285, Abcam, Cambridge, UK). After washing, the bound antibody was detected after incubation for 1 h, at room temperature, with the corresponding secondary antibody linked to the horseradish peroxidase enzyme. Quantification was performed using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/, 1997–2018, Accessed date 30 July 2021).

Antibodies against AIF, UBA52, Pink, Parkin, Tom20, Lamp2a, LC3B and α-SMA (Catalog numbers ab32516, ab109227, ab23707, ab77924, ab56783, ab18528, ab48394 and ab7817) were obtained from Abcam (Cambridge, UK). Antibodies against HK II, PKM2 and PDH (Catalog numbers 2867, 4053 and 3205) were obtained from Cell Signaling Technology (Danvers, MA, US). Antibodies against Cyclin A, Cyclin D, PCNA, UB, P62, ATG5 and ATG7 (Catalog numbers BM1582, BM4272, BM0104, BM4359, BA2849, BA3525 and BA3527) were obtained from Boster (Wuhan, China). Bortezomib (PS-341) and MG132 (Catalog numbers S1013 and S2619) were obtained from Selleck Chemicals (Pittsburgh, PA, US).

Total proteins were extracted from the realgar-treated NB4 cells, and the protein concentration was measured using a BCA Test Kit (Beyotime, China). The extracted proteins were denatured with reducing sample buffer at 100°C for 7 min. Then, 40 μg of protein was loaded onto a 10% polyacrylamide gel. Proteins were separated (30 min at 80 V, then 120 V until completed) with Tris-HCl running buffer in an electrophoresis apparatus (BioRad, Hercules, CA, USA). Then, the proteins were transferred (90 min, 100 V) from the acrylamide gel to a polyvinylidene fluoride (PVDF) membrane. Blots were blocked with 5% bovine serum albumin (BSA) for 1 h, followed by incubation with monoclonal anti-Bax (1:1000, ab32503, Abcam, USA), Bcl-2 (1:1000, ab32124, Abcam, USA), AIF (1:1000, ab32516, Abcam, USA), and Cyt-C (1:5000, ab133504, Abcam, USA) antibodies at 4°C overnight. Subsequently, the membranes were washed frequently with Tris-buffered saline Tween (TBS-T; 5 min, 3 times), followed by incubation with goat anti-rabbit IgG antibody (1:20,000) at room temperature for 1 h. After repeated washings with TBS-T, the blots were treated with a chemiluminescent detection kit (Thermo Fisher Scientific, China). The internal reference was represented by an antibody against actin. The quality of the protein was assessed using a Typhoon PhosphorImager (GE Healthcare, Piscataway, NJ, USA).

Deoxypodophyllotoxin (DPT) was obtained from Yuanye Bio-technology company (Shanghai, China). It was dissolved in DMSO and then stored at the concentration of 10 mmol/L. MnTBAP was from Sigma (St. Louis, MO, USA) and 3-Aminobenzamide (3AB), NAD⁺ and FK866 were all from MedChemExpress Company (Shanghai, China). The antibodies against TAX1BP1 (bs-13671R), NDUFS2 (bs-10455R) and NDUFS4 (bs-3961R) were all obtained from Bioss Antibodies (Beijing, China), against PARP1 (13371-1-AP), ND2 (19704-1-AP) and ND1 (19703-1-AP) were all from Proteintech Company (LA, USA), and against AIF (ab32516), GPX4 (ab125066), catalase (ab76024), H2AX (ab229914), TOMM20 (ab186735), phospho-H2AX at Ser139 (ab26350) and phospho-ATM at Ser1981 (ab81292) were all from Abcam company (Cambridge, UK), against PAR (#83732) and β-Actin (#4970) were both from Cell Signaling Technology Company (Beverly, MA, USA). Second antibody against rabbit (A0208) and against mouse (A0216) were from Beyotime Biotechnology (Nanjing, China).

U2OS cells were plated on coverslips coated with 10 μg/ml Poly‐d‐lysine (P6407‐5 mg Sigma) and 24 h later were left untreated or treated with 600 μM H₂O₂ for 9 h at 37°C. Cells were then washed once with ice‐cold phosphate‐buffered saline (PBS) and then fixed with ice‐cold methanol for 1 min. Coverslips were washed three times with PBS and then blocked with 3% BSA in PBS for 1 h at room temperature. Next, coverslips were incubated with primary antibodies (Rb α‐AIF, Abcam ab32516, 1:1,000 or Rb α‐NEDD8, Abcam ab81264, 1:1,000) diluted in PBS with 3% BSA for 1 h at room temperature. Coverslips were washed three times with PBS and then incubated with Alexa 488 conjugate anti‐rabbit secondary antibody (1:1,000, Thermo A‐11034) for 1 h at room temperature. DAPI (300 nM, Life Technologies, D1306) staining was performed for 5 min, followed by one wash with PBS. Coverslips were mounted onto slides with ProLong Gold Antifade reagent (Life Technologies) and imaged on a Zeiss LSM 710 confocal microscope. Quantification of nuclear AIF intensity was performed using ImageJ v1.50g (National Institute of Health).

For immunostaining, eyes were fixed in 4% paraformaldehyde (PFA) for 2 h, dehydrated in 30% sucrose, and embedded in OCT compound and snap frozen immediately. Sections (12 μm) were collected on a cryostat, dried at room temperature (RT) for 30 min, and fixed in 2% PFA for 10 min. After rinsing, the sections were blocked with 5% bovine serum albumin for 1 h at RT. The samples were incubated overnight at 4°C with specific primary antibodies: anti-cleaved caspase-3 [1:150; Cell Signaling Technology (CST), 9664], anti-rhodopsin (1:200; Millipore, MAB5316), anti-DCT (1:200; Bioworld, BS3320), anti-PEDF (1:100; Abcam, ab180711), anti-OTX2 (1:200; R&D Systems, AF1979) or anti-AIF (1:200; Abcam, ab32516). The staining was revealed by appropriate secondary antibodies [Alexa Fluor^® 488 donkey anti-rabbit IgG (H+L) (Life Technologies, A21206), Alexa Fluor^® 594 donkey anti-mouse lgG (H+L) (Life Technologies, A21203) and Alexa Fluor^® 488 donkey anti-goat IgG (H+L) (Life Technologies, A11055)]. Each staining was performed on slides from at least five animals per condition. Immunostaining results were observed and photographed on a Zeiss confocal microscope.