Nqdi 1

IMB-6G was synthesized according to previously described methods [18 (link)]. Antibodies against Bip, CHOP, PERK, IRE1α, eIF2α, phospho-PERK (Thr980), phospho-eIF2α (Ser51), phospho-ASK1 (Thr845), phospho-JNK (Thr183/Tyr185), Bax and Bad were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-phospho-IRE1α (Ser724) and ATF6 antibodies were from Abcam (Cambridge, MA, USA). Anti-Bim, PUMA and Cytochrome c antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). SP600125, NQDI-1, Thapsigargin, MTT and β-actin antibody were obtained from Sigma (St. Louis, MO, USA). Salubrinal (ER stress inhibitor) was purchased from Selleckchem (Shanghai, China).

Partial (70%) liver warm ischemia mouse model was established as previously described (Chen et al., 2021 (link)). Briefly, mice were first anesthetized by pentobarbital sodium (60 mg/kg; Sigma) and subjected to midline laparotomy. A microvascular clip was used to clamp the left and middle portal vein and hepatic artery branches to interrupt the blood supply of the liver. After 1 h of ischemia, the clamp was removed for reperfusion. After 6 h reperfusion, the animals were sacrificed to collect liver and serum samples for further analysis. The residual blood was discharged by portal vein injection of normal saline. Part of the liver tissue was stored in liquid nitrogen and then transferred to the refrigerator at −80°C. Another part of liver tissue was preserved in 10% formalin for pathological examination. As a sham control group, mice underwent the same surgical procedure but without vasculature clamping. To inhibit MIF and ASK1 in mice, specific ASK1 inhibitor NQDI-1 (Sigma; 10 mg/kg, dissolution by DMSO and dilute with PBS to 1.25 mg/ml, 200 μl per mice) and MIF inhibitor ISO-1 (MedChemExpress LLC; 3.5 mg/kg, dissolution by DMSO and dilute with PBS to 0.44 mg/ml, 200 μl per mice) were intraperitoneally injected 2 h before the ischemic surgery. The same volume of DMSO (diluted to 0.1% by PBS) was used as control (Xu et al., 2020 (link); Liu et al., 2021 (link)).

Adenoviruses encoding HDAC6 were generated using the pDC315‐Flag vector (GeneChem, Shanghai, China). For adenovirus‐mediated overexpression of HDAC6 in the retina, 8‐week‐old C57BL/6J mice were anaesthetized with 2% isoflurane, and one drop of 0.5% oxybuprocaine hydrochloride (Santen, Osaka, Japan) was applied to the surface of the cornea. The iris was dilated using 0.5% tropicamide phenylephrine (Santen), and mice were intravitreally injected with 1 µL solution containing 4 × 10¹⁰ PFU mL⁻¹ adenoviruses using a 34‐gauge Hamilton needle and syringe (Hamilton Company, Reno, NV) for the following 5 days.
For intravitreal injection of drugs, mouse eyes were treated using the above method. Tubastatin A (sml0044; Sigma‐Aldrich, St Louis, MO) was dissolved in DMSO (0.2 × 10⁻³ m) and diluted 1:10 with 0.9% saline; NQDI‐1 (sml0185; Sigma‐Aldrich) was dissolved in DMSO (1.2 × 10⁻³ m) and diluted 1:10 with 0.9% saline; cytochalasin D (ab143484; Abcam) was dissolved in DMSO (0.1 × 10⁻³ m) and diluted 1:10 with 0.9% saline; prostaglandin E2 (P0409; Sigma‐Aldrich) was dissolved in DMSO (0.5 × 10⁻³ m) and diluted 1:10 with 0.9% saline. Mice were treated by intravitreal injection with 0.5 µL of drugs or the same volume of vehicle every 2 days from P14, when they opened their eyes, until the study endpoint.

Mice partial liver ischaemia surgery was conducted as previously described.⁷ Mice were free to access to food and water for 12 hours before surgery. After 60 minutes of liver ischaemia by vascular occlusion, mice were reperfused and killed immediately (0 hours), 2, 6, 12 or 24 hours post‐reperfusion. Sham controls underwent the same operative procedure without vascular occlusion. Serum and tissue samples were collected for further analyses. We intraperitoneally administered the specific ASK1 inhibitor NQDI‐1 (Sigma; 10 mg/kg) to ARRB1 KO and WT mice 30 minutes before the ischaemic surgery. The same volume of DMSO was used as control.

Antibodies against HA, GST, GFP and Flag (Sigma-Aldrich, St. Louis, MO, USA); phosphoserine and phosphothreonine (Millipore, Billerica, MA, USA); CLIP-170, p150^glued and γ-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); α-tubulin (Abcam, Cambridge, MA, USA); pT845-ASK1 (Cell Signaling Technology, Danvers, MA, USA) and EB1 (BD Biosciences, San Jose, CA, USA) were purchased from the indicated sources. The anti-ASK1 antibody was obtained from Abcam (# ab45178). Horseradish peroxidase-conjugated secondary antibodies were from Amersham Biosciences (Chandler, AZ, USA). Rhodamine- or fluorescein-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). NQDI-1, paclitaxel and DAPI were from Sigma-Aldrich.

Antibodies against β-actin and α-tubulin (Sigma-Aldrich), ASK1 phosphorylated at threonine 845 (pASK1), ASK1, cyclin D, and cyclin E (Abcam), and horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) were obtained from the indicated sources. SRB, MTT, and NQDI-1 were purchased from Sigma-Aldrich. Control siRNA (5′-CGUACGCGGAAUACUUCGA-3′) and ASK1 siRNAs (#1: 5′-GCACUCCU-UCAUCGAGCU-3′; #2: 5′-GGUAUACAUGAGUGGAAUU-3′) were synthesized by Ribo Bio. The mammalian expression plasmid for HA-ASK1 was constructed by cloning ASK1 cDNA into the pCMV-HA vector. The HA-ASK1-KD mutant was generated by site-directed mutagenesis.