Ab144589

Human proximal tubular epithelial cells (HK-2 cells) were cryopreserved at the Institute of Kidney Disease, Central South University. SS31 was synthesised and provided by Chinapeptide Co. Ltd. (Shanghai, China). Streptozocin (STZ) was obtained from Sigma-Aldrich (USA). The selective Drp1 inhibitor Mdivi1 (ab144589) was obtained from Abcam (UK). Anti-fibronectin (FN) antibody (sc-52331), anti-Bcl-2 antibody (sc-56015), anti-IL-1β antibody (sc-52012), and anti-Bax antibody (sc-20067) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Drp1 rabbit monoclonal antibody (ab184247), anti-Mfn1 mice monoclonal antibody (ab57602), and Caspase1 antibody (ab138483) were purchased from Abcam (UK). The TUNEL assay kit (ab66110) and anti-β-actin antibody (ab8226) were purchased from Abcam (UK). Secondary antibodies in this study were purchased from KangChen Bio-tech (Shanghai, China). Other materials, including bovine serum albumin and low-glucose DMEM medium, were purchased from Gibco (USA).

E. hellem and E. cuniculi spores were gifted by Dr. Han Bing from Shandong University, China, and cultured in RK13 cells, respectively. One week after infection, spores were purified from culture media by centrifugation in 70% Percoll (17089102, Cytiva, Marlborough, MA, USA) at 10,000× g for 15 min at room temperature (RT), then washed three times with sterile water, suspended in 0.5 mL of sterile distilled water, and stored at 4 °C until use [14 (link),40 (link),42 (link)]. The HEK293 and HFF cells were inoculated with spores by a cell-to-parasite ratio of 1:30, and all samplings of the infected cells were prepared 48 h post-infection (hpi).
An inhibitor of DRP1, Mdivi1 (ab144589, Abcam), was added to HFF cells at a 50 μM concentration for 4 h prior to infection, and then replaced the fresh complete medium, following a previous study [27 (link)], and it was added again after 24 hpi and treated for 24 h. Additionally, the same concentration of dimethyl sulfoxide (DMSO) (D8418, Sigma Aldrich) was added as a control.

The experiments were divided into Vehicle group, H₂O₂ group, ZnG group, Mdivi‐1 group, or 3‐TYP group. To simulate SCI oxidative stress, VSC.4.1 was treated with H₂O₂.³⁶, ³⁷ H₂O₂ treated VSC4.1 with H₂O₂ (80 μmol/L) for 3 h. The ZnG group was treated with H₂O₂ (80 μmol/L) for 3 h, followed by ZnG (100 μmol/L, # Z820656‐100 g, Macklin) for 24 h. The Mdivi‐1 group was treated with the mitophagy inhibitor Mdivi‐1 (25 μmol/L, # ab144589, Abcam) for 3 h before the treatment with the addition of H₂O₂, and the rest of the treatment was the same as the ZnG group.³⁸ The 3‐TYP group was treated with the SIRT3 inhibitor 3‐TYP (50 μmol/L, # IT1960, Solarbio) before adding H₂O₂ for 3 h, and the rest of the treatment was the same as that of the ZnG group.³⁶ The Vehicle group was cultured in DMEM, and other treatment factors were identical.