Cleaved caspase 3

Total proteins were extracted from cells a using RIPA buffer (Beyotime, Shanghai, China). Then, protein samples (30 μg per lane) were separated on 10% SDS-PAGE gels (Bio-Rad, USA) and transferred to a PVDF membrane (Millipore, USA). Sealed with PBS buffer containing 5% skim milk powder for 2 h, and added anti-Bcl-2 (1: 1000, Beyotime, Shanghai, China), anti-Bax (1∶2000, Beyotime, Shanghai, China), anti-cleaved caspase3 (1∶2000, Beyotime, Shanghai, China), anti-GAPDH (1∶2000, Beyotime, Shanghai, China) overnight at 4 °C, the PBST buffer was rinsed 3 times, 5 min each time. Next, Bcl-2, Bax, cleaved caspase3 and GAPDH secondary antibodies (sheep versus rabbit, Beyotime, Shanghai, China) were added, and incubated at 37 °C for 4 h. PBST buffer was rinsed for 3 times, and the gray value of target protein was analyzed by Image J.

Sodium hydrogen sulfite and sodium sulfite (NaHSO₃/Na₂SO₃, freshly mixed at 1:3 M ratio, pH 7.4) were used as SO₂ donors and purchased from Sigma (Zhang et al., 2021 (link)). Isoprenaline hydrochloride was purchased from Sigma (I5627). A chemically selective fluorescent probe SS-1 was provided by Professor Kun Li and Xiaoqi Yu. DAz-2 was used as a protein sulphenylation probe (13382, Cayman, Michigan, USA) to capture and enrich the sulphenylated protein. The primary antibodies in the present study included CypD (abcam, USA), AAT1 (Sigma, USA), AAT2 (Sigma, USA), β-actin (Zsbio, China), caspase9 (CST, USA), caspase3 (Beyotime, China), cleaved caspase3 (Beyotime, China), cytc (santa, USA), His (Zsbio, China), and β-tubulin (Zsbio, China). Human CypD wild type (WT), and C104S, C82S, C157S, and C203S mutant plasmids were constructed by Sangon Biotech. The information of three kinds of ATP plasmids used in this study are as follows: EcAT3.10 (Conley et al., 2017 (link)) was deposited at Addgene by Mathew Tantama (Addgene plasmid #107215); pm-iATPSnFR1.1 (Lobas et al., 2019 (link)) (Addgene plasmid #102549) and cyto-iATPSnFR1.0 (Lobas et al., 2019 (link)) (Addgene plasmid #102550) were deposited at Addgene by Baljit Khakh.

The radioimmunoprecipitation assay buffer (RIPA, Beyotime, China) was employed for extracting total cellular proteins in THCA cells after transfection. WB assay was carried out to measure protein levels according to previous description. This work acquired primary antibodies against siglec-15 (1 : 500; Abcam), VEGF (1 : 800; Abcam), STAT1 (1 : 500; Beyotime), STAT3 (1 : 500; Beyotime), GAPDH (1 : 500; Beyotime), and cleaved caspase-3 (1 : 500; Beyotime), together with relevant secondary antibody (1 : 500) in Santa Cruz Biotechnology (Shanghai, China), with GAPDH being the endogenous reference. Odyssey was employed for band detection, whereas Image Studio Software (LI-COR Bioscience, Lincoln, Nebraska, USA) was utilized in band analysis.

Proteins of the neurons were extracted by a RIPA lysis buffer (#P0013C, Beyotime Biotechnology) containing 1% phenylmethylsulfonyl fluoride (PMSF, Roche), followed by centrifuging at 4 °C, 13,000 rpm, 25 min to collect the supernatant. Proteins were denatured and separated by 10% SDS-PAGE, transferred to PVDF (polyvinylidene fluoride) membrane. The primary antibodies were used as follow: Anti BDNF (#AF1423), total-Akt (#AF0045), phospho-Akt (Ser473) (#AF1546), Bcl-2 (#AF0060), β-actin (#AF0003) and cleaved-Caspase-3 (#AF1150) were provided by Beyotime Biotechnology. Anti phospho-TrkB (#4619) antibody was provided by Cell Signaling Technology, Inc. The secondary antibodies including: Goat anti-Mouse IRDye 800CW (#926-32210), Goat anti-Rabbit IRDye 800CW (#926-32211), IRDye 680RD Goat anti-Mouse (#926-68070) and IRDye 680RD Goat anti-Rabbit (#926-68071) were provided by Beijing North Yitao Trading Co., Ltd. Western blot images were scanned by the Odyssey CLx infrared fluorescence imaging system (LI-COR Biosciences). The normalized protein expression level was calculated according to the optical densities of the blots that were quantified by Image J software.

Western blotting was conducted with primary antibodies against PRDX3 (Abcam, Ltd., Cambridge, UK), SIRT3 (Cell Signaling Technology, MA, USA), cleaved caspase-3 (Beyotime Institute of Biotechnology, Shanghai, China), TOMM20 (Cell Signaling Technology, MA, USA) and β-actin (Beyotime Institute of Biotechnology, Shanghai, China). Protein quantification was performed using Gel-Pro Analyzer version 4.0 (Media Cybernetics, MD, USA).

The human bladder cancer cell lines 5637 and T24 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Both of them were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, glutamine, and antibiotics at 37°C in 5% CO₂.
p-Akt and Akt antibodies were bought from Cell Signaling Technology (USA). Antibodies to β-actin, Bcl-2, mcl-1, and cleaved caspase-3, as well as horseradish peroxidase- (HRP-) linked anti-rabbit IgG, were purchased from Beyotime Institute of Biotechnology (China). MTT were purchased from Sigma (USA). MTT, Annexin-V-FITC, and PI were purchased from Beyotime Institute of Biotechnology, respectively. miR-155 mimics and inhibitor or negative siRNA control, as well as the primers for U6 and miR-155, was brought from Qiagen (Germany).