Tubastatin a

For determination of α-tubulin acetylation and nuclear TFEB, NRK-52E cells (Advani et al., 2009 (link)) were treated with Tubastatin A in 0.1% DMSO for 24 h. For determination of acetylated TFEB levels, NRK-52E cells were treated with 2.5 μM Tubastatin A (Butler et al., 2010 (link)) (or vehicle) for 24 h prior to immunoprecipitation for TFEB (Abcam, Cambridge, MA, United States). For determination of ER stress in NRK-52E cells, cells were treated with 500 nM thapsigargin (Sigma–Aldrich, Oakville, ON, Canada) for 24 h. For determination of programmed cell death, NRK-52E cells were pre-treated with 2.5 μM Tubastatin A (or vehicle) for 4 h, before the addition of 500 nM thapsigargin for a further 24 h. For annexin V labeling, following labeling with PE-annexin V 7-aminoactinomycin D (7-AAD) (BD Biosciences, San Jose, CA, United States) cellular fluorescence was assessed on a MACSQuant Analyzer (Miltenyi Biotec, Cambridge, MA, United States). For gene knockdown, cells were transfected with sequence-specific siRNA or scrambled siRNA (Thermo Fisher Scientific, Rockford, IL, United States) at concentrations of 75 nM for HDAC6 and 50 nM for TFEB for 24 h using Lipofectamine 2000 (Thermo Fisher Scientific).

Following the SCI procedure, mice in the Tubastatin‐A group were intraperitoneally injected with Tubastatin‐A (Yuanye, S80226) at 50 mg/kg/day,
²⁰ (link) while mice in the model and sham groups were injected with the same amount of saline solution. For the cellular experiment, MECs from the Tubastatin‐A group were treated with 40 μM Tubastatin‐A, while MECs of the Baf‐A1 group were treated with 50 nM bafilomycin A1 (Abcam, ab120497).
¹⁶ (link) These indices were measured after 72 h.

Chemicals were purchased from Sigma–Aldrich Company (St. Louis, MO, USA) unless otherwise indicated. The HDAC inhibitor trichostatin A (TSA) (ab120850) and HDAC6 inhibitor tubastatin A (ab141415) were purchased from Abcam (Cambridge, MA, USA). The primary antibodies used were rabbit anti-HDAC6 (cat. 7558, Cell Signaling (Danvers, MA, USA), 1:1000 for WB, 1:200 for staining), rabbit anti-GAPDH (cat. 5174, Cell Signaling, 1:10000 for WB), rabbit anti-Ac-K (cat. 9441, Cell Signaling, 1:1000 for WB), rabbit anti-Myc (cat. PA9064, Abmart, 1:1000 for WB), rabbit anti-Flag (cat. F7425, Sigma, 1:2000 for WB), mouse anti-NeuN (cat. MO22122, Neuromics (Edina, MN, USA), 1:1000 for staining), rabbit anti-GFAP (cat. Z0334, Dako (Kyoto, Japan), 1:1000 for staining), rabbit anti-Iba1 (cat. Ab5076, Abcam, 1:500 for staining), rabbit anti-γH2AX (cat. 9718, Cell Signaling, 1:1000 for WB, 1:500 for staining), and MIF (cat. Ab187064, Abcam, 1:1000 for WB, 1:500 for staining). The MIF K78 acetylation antibody was generated by ABclonal Biotechnology (Shanghai, China).