U0126

CRLF1-overexpressing IHH-4 cells were grown as described above until they reached 40% confluence. Then, the media were replaced with RPMI-1640 containing vehicle (0.1% (v/v) DMSO), U0126 (10 μM) (S1102, Selleck, Houston, TX), MK-2206 (10 μM) (S1078, Selleck, Houston, TX), or U+M (10 μM U0126+10 μM MK-2206). Samples were collected at different time intervals as indicated for each experiment.

The human pancreatic cancer cell lines PANC-1, SW1990, ASPC1, BXPC3, and CFPAC1 were obtained from the Type Culture Collection Committee of the Chinese Academy of Sciences. All cells were maintained in RPMI 1,640 medium, DMED, or IMDM supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. For hypoxia induction, a hypoxia-mimetic agent, cobalt chloride (CoCl₂), dissolved in distilled water, was added to cell cultures at the indicated final concentrations for 24 h, whereas water was added to the culture medium alone as a vehicle control. To inhibit and activate the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway, we used the ERK inhibitor U0126 and the ERK1/2 activator senkyunolide I (SENI), which were purchased from Selleckchem (U0126: SCH772984; SENI: S3275) and dissolved in dimethyl sulfoxide. The cells were pretreated with U0126 (10 μM), SENI (5 μM), or CoCl₂ for 24 h.

Peritoneal macrophages were prepared as described previously (Zhang et al. 2021b (link)) and were cultured in RPMI 1640 medium supplemented with 10% FBS (SH30071.03, HyClone, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin. 2 μg/mL of LT [2 μg/mL of LF (172C, List Biological Labs) and 2 μg/mL of PA (171E, List Biological Labs)] was used to treat peritoneal macrophages for 3 h. HT-29 cells of indicated genotypes were cultured in DMEM supplemented with 10% FBS (10099-141, Gibco, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin. 2 μg/mL of LT, 30 ng/mL of human TNF (PHC3011, Gibco), and 10 μmol/L of LCL-161 were used to induce cell death in HT-29 cells. Inhibitors used in this study were 2 μmol/L of SB202190 (S1077, Selleck, USA), 2 μmol/L of SB203580 (559389, Calbiochem), 2 μmol/L of TAK-715 (HY-10456, MedChemExpress), 2 μmol/L of U0126 (S1102, Selleck), 2 μmol/L of GDC-0994 (S7554, Selleck), and 10 μmol/L of SP600125 (S5567, Sigma–Aldrich, USA).

α-Hederin of purity over 99% was provided by Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). NF-κB specific inhibitor pyrrolidine dithiocarbamate (PDTC), JAK2/STAT3 signaling specific inhibitor AG490, and ERK specific inhibitor U0126 were obtained from Selleck Chemicals (Houston, TX, USA). Dimethyl sulfoxide (DMSO) was used to dissolve the above compounds for experiments, and single treatment with DMSO was used as negative control. Recombinant human IL-6 cytokine was obtained from Solarbio Life Science (Beijing, China). The primary antibodies used for Western blot analyses against cyclin B1, CDK1, Bcl-2, Bax, Cyt c, cleaved-caspase-9, cleaved-caspase-3, cleaved-caspase-8, cleaved-PARP, NF-κB(p65), p-IκBα, IκBα, p-IKKα, IKKα, p-ERK, ERK, COX IV, lamin B1, and GAPDH were purchased from Bioworld Technology, Inc. (MN, USA).

Male mice in 8–12-week-old were anesthetized for 1 min with 2% isoflurane in oxygen, and the maintenance dose during the operation was 1% isoflurane in oxygen. After disinfection and depilation in the left lower limbs of the mice, the left femoral artery was isolated by blunt dissection under a surgical microscope. The distal artery was looped with a 6–0 nylon suture, and a 0.38 mm guide wire (Cook Inc, Bloomington, IN) was inserted into the arterial lumen through an arteriotomy in the distal perforating branch. The wire was left in the artery for 2 min to denude the artery and was carefully removed. The right femoral artery was used as the sham group. The nylon suture was released to restore the blood flow, and the opening was sutured with a 6–0 nylon suture. The mice were placed on a heating blanket until recovery, and the femoral arteries were collected after 28 days.
The mouse femoral artery injury model was also performed with or without the U0126 treatment. U0126 was purchased from Selleck (S1102) and dissolved in dimethyl sulfoxide (DMSO) to a concentration of 0.1 mg/mL. It was injected intraperitoneally into mice every 3 days (1 mg/kg) after wire injury. The dose of U0126 was determined based on a previous study [18 (link)]. The control group was injected with a similar volume of DMSO.

d-Pantethine, imipramine, GW4869, calpeptin, Y-27632, imatinib mesylate, sulfisoxazole, bisindolymaleimide I, indomethacin, NSC23766, clopidogrel, glibenclamide, Chloramidine, amiloride, and U0126 were purchased from Selleckchem (Selleckchem, Houston, TX, USA). Manumycin A and cytochalasin D were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Luis, MO, USA). All compounds (purity > 97%) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Luis, MO, USA), at a concentration of 50 mM, before use.

CCK‐8 kit and Annexin V‐FITC/PI Apoptosis Detection kit were purchased from Dojindo (Kumamoto, Japan). PI, Glutaraldehyde solution, 2′, 7′‐Dichlorodihydrofluorescein diacetate (DCFH‐DA) and N‐acetyl‐L‐cysteine (NAC) are from Sigma‐Aldrich (St Louis, MO, USA). 3‐Methyladenine (3‐MA), Bafilomycin A1 (Baf‐A1), Dorsomorphin (Compound C), SB202190, U0126 and SP600125 were obtained from Selleck (Houston, Texas, USA).4′,6‐diamidino‐2‐phenylindole (DAPI), Lyso‐Tracker Red and Lipid Peroxidation MDA Assay kit were purchased from Beyotime (Nantong, China).
Antibodies against Cyclin B1 (ET1608‐27), Cyclin A2 (M1511‐5), CDK2 (ET1602‐6), CDK1 (ET1605‐54), ATG5 (ET1611‐38), p38 (ET1602‐26), ERK1/2 (ET1601‐29), phospho‐ERK1/2(Thr202) (ET1610‐13), JNK1/2/3 (ET1601‐28), phospho‐JNK1/2/3(T183 + T183 + T221) (ET1609‐42), AMPK alpha 1 (ET1608‐40), phospho‐AMPK alpha 1 (S496) (ET1612‐72), HSPB1 (ET1701‐70), phospho‐HSPB1(S82) (ET1611‐16) and PARP (ET1608‐56) were purchased from HuaBio (Hangzhou, China). Antibodies against β‐actin (66009‐1‐lg), p53 (10442‐1‐AP), p21 (10355‐1‐AP), p62 (18420‐1‐AP), BAX (50599‐2‐1g) and caspase 3 (19677‐1‐AP) were obtained from Proteintech (Wuhan, China). The antibodies against phospho‐p38 (T180/Y182) (9211S) and LC3B (2775s) were purchased from Cell Signaling Technology (Beverly, MA, USA).