B2064

The morphology of the oocyte spindles and chromosomes was evaluated by immunofluorescence staining. First, the MII oocytes were fixed in 4% PFA for 30 min at 4 °C. Then, oocytes were permeabilized in 5% Triton X-100 (X-100, Sigma-Aldrich) for 20 min and blocked in 3% (w/v) bovine serum albumin (BSA; B2064, Sigma-Aldrich) for 1 h at room temperature. Next, oocytes were incubated with monoclonal anti-β-tubulin antibody (SAB4200715, Sigma-Aldrich) (1:500 dilution) for 24 h at 4 °C. Then, the oocytes were incubated in Alexafluor 488 rabbit anti-mouse IgG (H + L) secondary antibody (A-11059, Invitrogen) (1:500 dilution) for 1 h at room temperature after washing three times with blocking solution (5 min each time). Then, the DNA was stained with 5.0 μg/mL Hoechst 33342 solution (C0030, Solarbio, China) for 20 min and washed three times with PBS (3 min each time). Finally, images of oocytes were taken by a confocal laser scanning microscope (C2 Plus, Nikon, Japan).

Both cells and liver tissue sections were fixed with 4% paraformaldehyde for 15 minutes (P6148, Sigma–Aldrich), followed by permeabilization with 0.3% Triton X-100 (1139ML100, Biofroxx) for 5 minutes. After three PBS washes, the cells were blocked in 4% bovine serum albumin (BSA; B2064, Sigma–Aldrich), and the liver tissues were blocked with 5% goat serum (S9070, Solarbio) for 30 minutes. The samples were then incubated overnight with the target antibody at 4 °C. After another three PBS washes, the cells were treated with Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies (A21202, A10042, A31573, Thermo Fisher Scientific) for 1 hour, followed by DAPI staining (D212, Dojindo). The primary antibodies used included anti-TXNIP (Huabio, ET1705-72, 1:200) and anti-Tomm20 (Santa Cruz Biotechnology, sc-17764, 1:200). Fluorescence images for every sample (200× magnification or 400× magnification) were captured using a fluorescence microscope (IX81-FV1000, Olympus). The antibodies used for immunofluorescence are listed in Table S2.

The protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane, followed by blockage with 5% bovine serum albumin (Sigma, B2064) in Tris-buffered saline (Sigma, T5030) containing 0.1% Tween 20 (Sigma, 93773) (TBST). The membranes were then incubated with primary antibodies at 4°C overnight. The primary antibodies included anti-NF-κB p65 (Abcam, ab16502), anti-Nrf2 (Abcam, ab31163), anti-GAPDH (glycer-aldehyde-3-phosphate dehydrogenase) (Abcam, ab9485), and anti-Lamin B1 (Proteintech, 66095-1-Ig) antibodies. After three washes with TBST, the membranes were incubated with HRP-goat-anti-rabbit (Abcam, ab6721) or HRP-goat-anti-mouse (Abcam, ab6789) secondary antibodies at room temperature for 1 h, followed by visualization using an enhanced chemiluminescence (ECL) system (Thermo Scientific, 32132). The images of protein bands were photographed using a ChemiDoc MP device (Bio-Rad, Hercules, CA, USA) and analyzed using ImageJ software.

After specific treatment, cultured cells grown on 96‐well plate were washed with PBS twice and incubated with 4% paraformaldehyde (P6148, Sigma‐Aldrich, St. Louis, USA) for 20 min. Then, the cells were permeabilized with the treatment of 0.1% Triton X‐100 in PBS for 10 min at 4 °C and blocked with 4% bovine serum albumin (B2064, Sigma‐Aldrich, St. Louis, USA) in PBS for 0.5 h at 37 °C. The primary antibody was added and incubated with the cells at 4 °C overnight. At the end of incubation, the primary antibody was removed and the cells were washed with PBS. Then cells were incubated with Alexa Fluor 488‐ or Alexa Fluor 568‐conjugated secondary antibodies (A11008, A10037; 1:100, Thermo Fisher Scientific, Waltham, USA) at room temperature for 1 h. Nuclei were stained with DAPI for 5 min and then imaged with a fluorescence microscope (IX81‐FV1000, Olympus, Tokyo, Japan). The following primary antibodies were used: anti‐Bcl‐XL (ET1603‐28, Huabio, Hangzhou, China), anti‐USP13 (sc‐514416, Santa Cruz Biotechnology, Dallas, USA).

Oocytes were fixed for 30 min in PBS containing 2% formaldehyde and 0.05% Triton X-100 and then permeabilized in 0.5% Triton X-100 for 20 min at room temperature. Oocytes were incubated at 4 °C overnight in a blocking buffer with 3% bovine serum albumin (B2064, Sigma-Aldrich) in PBS supplemented with 0.1% Tween-20 and 0.01% Triton X-100. Oocytes were incubated at 37 °C with following primary antibodies: anti-β-tubulin antibody (1:200 dilution, ab11309, Abcam), anti-FLAG antibody (1:50 dilution, ab245893, Abcam), anti-centromere antibody (ACA) (1:500 dilution; HCT-0100, ImmunoVision), anti-HEC1 phospho Ser55 antibody (1:200 dilution, GTX70017, GeneTex), anti-BUBR1 antibody (1:100 dilution, 612502, BD Biosciences), and anti-KIF11 antibody (1:200 dilution, HPA010568, Sigma-Aldrich). After washing, oocytes were incubated at 37 °C for 1 h with appropriate secondary antibodies as follows: goat anti-human Alexa Fluor 488 (1:500 dilution, 52526, Sigma-Aldrich), goat anti-rabbit Cy3 (1:500 dilution, AS007, ABclonal), and goat anti-rabbit Alexa Fluor 647 (1:500 dilution, AS060, ABclonal). DNA was counterstained with Hoechst (5 μg/mL, 1:1000 dilution, HY-15559A, MCE) for 10 min at room temperature before imaging. Finally, the oocytes were mounted on a 35-mm glass-bottom dish (801001, NEST) and images were captured using a confocal laser-scanning microscope (Leica SP8 or ZEISS LSM 880).