Df7198

We used the same method as before to perform Western blotting.³⁷ Before Western blotting, we used a total protein extraction kit (BC3710, Solarbio), cellular mitochondrial isolation kit (C3601, Beyotime) or tissue mitochondrial isolation kit (C3606, Beyotime), and nuclear protein extraction kit (R0050, Solarbio) to extract total protein, mitochondrial protein or de‐mitochondrial cytoplasmic protein, and nuclear protein, respectively. The primary antibodies used were anti‐PARP‐1 (1:1000, DF7198, Affinity); anti‐AIF (1:1000, BF0591, Affinity); anti‐COX IV (1:1000, AF5468, Affinity); anti‐Histone H3 (1:1000, AF0863, Affinity); anti‐β‐actin (1:10,000, 66009‐1‐Ig, Protentech); anti‐LC3BI/II (1:1000, A5402, Affinity); anti‐PINK1 (1:200, PA5‐85930, Invitrogen); anti‐Parkin (1:200, 702785, Invitrogen); anti‐SIRT3 (1:1000, Affinity, AF5135); anti‐SOD2/MnSOD (acetyl K68) (1:5000, ab137037, Abcam); and anti‐SOD2/MnSOD (1:5000, ab13533, Abcam). The second antibodies used were HRP‐conjugated AffiniPure Goat Anti‐Mouse IgG (1:10,000, Proteintech) and HRP‐conjugated AffiniPure Goat Anti‐Rabbit IgG (1:10,000, Proteintech). Finally, PVDF molds were visualized on a Tanon 2500R gel imaging system (Tanon) using a developer (Tanon), and the band intensity was quantified using ImageJ 1.39V software.

VSC4.1 neurons or spinal cord frozen sections were fixed using 4% paraformaldehyde, then incubated with 0.3% Triton X‐100 for 10 min, bovine serum albumin (BSA) for 2 h, then incubated with primary antibody overnight at 4°C and with secondary antibody at room temperature for 2 h. In the end, the sections were blocked by incubating with DAPI at room temperature for 10 min. The primary antibodies are as follows: anti‐SIRT3 (1:500, AF5135, Affinity); anti‐PARP‐1 (1:500, DF7198, Affinity); anti‐AIF (1:500, BF0591, Affinity); anti‐NeuN (1:1000, ab104224, Abcam); anti‐β‐tubulin (1:500, 66240‐1‐Ig, Proteintech) The second antibodies are as follows: Alexa Fluor 488/568 goat anti‐mouse IgG, Alexa Fluor 568 goat anti‐rabbit IgG (1:1000, Thermo Fisher Scientific).

Following drug treatment (BMN673: 1 μM, JQ1: 1 μM), the cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Total proteins were quantified using a BCA Protein Assay Kit, separated using SDS-PAGE, then transferred to PVDF membranes. Antibodies against the following proteins were used for our studies: c-MYC (1:1,000, abcam ab32072), PARP1 (1:1,000, Affinity DF7198), GAPDH (1:10,000, Affinity AF7021), Rad51 (1:10,000, Abcam ab133534), PARP (1:1,000, CST 9542), γH2AX (1:1,000, CST 2577), p-CHK1 (1:1,000, Ser317, CST 12302), BRD2 (1:1,000, Proteintech 22236-1-AP), BRD3 (1:1,000, Proteintech 11859-1-AP), BRD4 (1:500, Bethyl A301-985A50), p-DNA-PKcs (1:1,000, Abcam ab124918), p-RPA32 (1:5,000, Ser4/Ser8, Novus), MYCN (1:1,000, CST 9405), MYCL (1:1,000 R&D, AF4050) and β-actin (1:10,000, Transgen HC201-02). Rabbit IgG (1:10,000, CST 7074) and mouse IgG (1:10,000, CST 7076) were used as secondary antibodies.