Sb203580

Thymoquinone, bovine serum albumin (BSA) and the antibody against alpha‐smooth muscle actin (α‐SMA) were obtained from Sigma. Thymoquinone was stored at 4°C and dissolved in olive oil. Foetal bovine serum (FBS) and DMEM were obtained from Life Technologies. The antibodies against cleaved PARP, Bax, voltage‐dependent anion channel (VDCA), cytochrome C, p‐c‐Jun NH2 terminal protein kinase (p‐JNK), JNK, p‐p38MAPK and p38MAPK were purchased from Abcam. The antibodies against cleaved caspase 3, p‐AKT, AKT, p‐extracellular signal‐regulated kinase 1/2 (p‐ERK), ERK1/2, Bcl‐2, GAPDH, β‐actin, the peroxidase‐conjugated anti‐rabbit and anti‐mouse secondary antibodies were purchased from Cell Signaling Technology. The antibody against PTEN was purchased from Santa Cruz). SB203580 (a p38MAPK inhibitor) was purchased from TargetMol. MTT kit was purchased from Beyotime Biotechnology.

Isotalatizidine was extracted and purified by the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, and the purity was validated as > 95% using high-performance liquid chromatography. For the experiments, it was dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted in sterile saline (0.9%). SB203580 and U0126-EtOH were all purchased from TargetMol (Shanghai, China). KG-501 was purchased from MedChemExpress (Shanghai, China) and dissolved in DMSO, and diluted with DMEM, DMEM/F-12, or saline before use. The cell culture reagents were purchased from the Invitrogen Corporation (Thermo Fisher Scientific, Carlsbad, CA, USA). Anti-dynorphin A antibody was purchased from Abcam (Cambridge, UK), and the Alexa 546-conjugated goat anti-rabbit and Alexa 488-conjugated goat anti-mouse secondary antibodies from the Life Technology (Thermo Fisher Scientific, Carlsbad, CA, USA). The remaining antibodies were purchased from the Cell Signaling Technology (Beverly, MA, USA). The goat serum was purchased from the Beyotime Biotechnology (Shanghai, China), and triton X-100 from the Sigma Aldrich.

Antibodies against the following proteins were used for Western blot: RPS15 (1:2000, Abcam; ab157193), IGF2BP1 (1:1000, Cell Signaling Technology; 8482), phospho-p38 MAPK (1:1000, Cell Signaling Technology; 9211), p38 MAPK (1:1000, Cell Signaling Technology; 9218), MKK6 (1:1000, Cell Signaling Technology; 8550), β-actin (1:1000, Cell Signaling Technology; 4970), Flag-tag (1:5000, Immunoway; YM3001), Myc-tag (1:5000, Immunoway; YM3203), m⁶A antibody (Thermo; MA5-33030) and rabbit IgG (Millipore; PP64B)-Diquafosol tetrasodium (CAS: 211427-08-6; T7423), Coenzyme A (CAS: 85-61-0; T10857), Folic acid (CAS: 59-30-3; T0062), Epicatechin gallate (CAS: 1257-08-5; T2732), and Methotrexate (CAS: 59-05-2; T1485) were purchased from TargetMol and SB203580 (CAS: 152121-47-6; T1764).