Tunicamycin

Intrathecal injection was performed as described previously (Liu Y.D. et al., 2019 (link)). Briefly, laminectomy was performed at the L5 vertebra under anesthesia. PE-5 catheter was inserted into the subarachnoid space of the spinal cord at L4 level. The rapamycin (autophagy inducer, CST, 0.2 μg/10 μl), 3-MA (autophagy inhibitor, Selleck, 50 μM), 4-PBA (ER stress inhibitor, Selleck, 200 μM), and tunicamycin (ER stress inducer, Selleck, 25 μM) were intrathecally administered 10 μl on operation day and postoperative day 2, 4, 6, 8, 10, 12, and 14 in SNL + RAP, SNL + 3-MA, SNL + 4-PBA, and SNL + TM group, respectively. All the groups without specific noted harvested spinal cord (L3–L5) at postoperative 14 day. The sham + C group received sham operation and catheter implantation procedure. SNL + C group received spinal nerve ligation and catheter implantation. The sham + C and SNL + C groups received 10 μl saline via catheter on operation day and postoperative day 2, 4, 6, 8, 10, 12, and 14.

The following antibodies were used in this study: FBXO2 (sc-398111, Santa Cruz), EBNA1 (ab81581, Abcam), Zta (sc-53904, Santa Cruz), β-tubulin (sc-23948, Santa Cruz), Myc (sc-40, Santa Cruz), GAPDH (60004-1-1g, Protein-tech) and FLAG (F3165, Sigma). MG132, bafilomycin A1 and tunicamycin were purchased from Selleckchem, LLC. siRNAs were synthesized by Guangzhou RiboBio Co., Ltd. The siRNAs were 21 base pairs long, and the sequences were as follows: control siRNA: UUCAAUAAAUUCUUGAGGUdTdT; FBXO2 siRNAs: #1: GAUGAGAGCGUCAAGAAGUdTdT; #2: CAGUUCUACUUCCUGAGCAdTdT; #3: AAGGUAGAUAGGCCUUAACdTdT. Lipofectamine RNAiMAX (Invitrogen) was used for siRNA transfection.

Dr. F. van Valen (Münster, Germany) kindly provided WE-68 cells. A673 cells were purchased from Sigma Aldrich and SaOS-2 cells were purchased from the DSMZ. HCT116 p53^wt and p53^−/− colon cancer cells were a gift from Dr. B. Vogelstein (Baltimore, MD, USA). WE-68 cells were maintained in RPMI 1640, SaOS-2 cells were maintained in McCoy's 5A medium, A673 and HCT116 cells were maintained in DMEM with 4.5 g/l glucose (all from Thermo Fisher). RPMI 1640 and DMEM were supplemented with 10% FCS and McCOY's 5A was supplemented with 15% FCS; all media were supplemented with 2 mM L-glutamine and 100 U/ml penicillin/streptomycin (all from Thermo Fisher). ES cells were cultivated in collagen-coated (5 μg/cm²; Thermo Fisher) tissue culture flasks. Dr. A. Poth (Roßdorf, Germany) kindly provided BALB/c-3T3-A31-1–1 cells from Hatano Research Institute of Japan. BALB/c cells were maintained in DMEM/HAM's F-12 (3.0 g/l glucose; Biochrom) supplemented with 5% FCS and 100 U/ml penicillin/streptomycin. Only sub-confluent cells (about 70% confluence) between the passages 20 to 40 were used for the BALB-CTA. All cells were cultivated in a humidified incubator at 37 °C with 5% CO₂.
Cells were treated with 0–50 mM AUY922 (Luminespib; S1069), 1–10 µM VE821 (S8007), 5–15 µM KU55933 (S1092), 0.4–5 µg/ml tunicamycin (S7894) (all in DMSO and from Selleck Chemicals) or their combinations for up to 72 h.