Sp600125

PI3K inhibitor LY294002, ERK1/2 inhibitor SCH772984, JNK inhibitor SP600125, p38 inhibitor SB203580, capmatinib and defactinib were purchased from MedChemExpress. The agents were used under the standard protocols. The HCC cells were pretreated with PI3K inhibitor LY294002 (10 μM), ERK1/2 inhibitor SCH772984 (10 μM), JNK inhibitor SP600125 (10 μM), or p38 inhibitor SB203580 (10 μM) for 1 h. defactinib was orally administered at a dose of 25 mg/kg twice a day, capmatinib was orally administered at a dose of 10 mg/kg/day. The treatment began on the seventh day after the establishment of the animal model and lasted eight weeks.

Oleic acid (OA) (Sigma-Aldrich) was mixed with 3 ml of 0.1 mM NaOH (Shanghai Macklin Biochemical Co., Ltd, Shanghai, China) and then saponified at 75 °C for 30 min. Next, the OA solution was mixed with 20% bovine serum albumin (BSA) (MRC, Shanghai, China), at 55 °C for 30 min. The stock solution of OA at 10 mM in 10% BSA was then used to make the working solutions.
Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich. Bay-11-7082, SP600125, U0126, and SB203580 were purchased from MedChemExpress LLC (Monmouth Junction, NJ, USA).

Curcuma Longa Linn (powdered form) and lecithin (L-α-phosphatidylcholine) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The organic solvents such as toluene and dichloromethane were purchased from Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from GE Healthcare (Logan, UT, USA). The following antibodies were obtained: c-Src, phospho-c-Src, PKC, phospho-PKC, JNK, phospho-JNK, p38, phospho-p38, ERK, phospho-ERK, IκBα, phospho-IκBα, NF-κBp65, phospho-NF-κBp65, Bcl-2, Bax, cleaved caspase-3, and β-actin antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA); The following reagents were obtained: N-acetylcysteine (NAC) (Tocris, KOMA Biotech, Seoul, Korea) and 5-(and-6)-chloromethyl-2′,7′- dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) (Invitrogen, Carlsbad, CA, USA). PP2, SP600125, Bisindolylmaleimide I, and Bay11-7082 were obtained from MedChemExpress (Monmouth Junction, NJ, USA). All other reagents did not show any critical cytotoxic effects by themselves.

Human ER-positive breast cancer cell lines MCF7 and T47D (ATCC), and HEK293T (ATCC) cells were cultured in DMEM (Hyclone) with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Hyclone) at 37 °C in a humidified 5% CO₂ atmosphere. Tamoxifen-resistant (TamR) MCF7 and T47D cells were generated as described previously [63 (link)]. In brief, cells were cultured in the presence of increasing concentrations of 4-hydroxytamoxifen (Sigma-Aldrich) starting at 0.5 μM, and finally gradually increased up to 5 μM 4-hydroxytamoxifen when the growth of the cells could not be inhibited in this concentration. In parallel, parental MCF7 and T47D cells were cultured under identical conditions without tamoxifen. SP600125, etomoxir and puromycin was obtained from MedChem Express.

Cytokines and Hyaluronan (HA) were assessed in triplicate by ELISA. TAO-OFs were treated with either PBS, IFN-γ (100 U/mL; Sino Biological Inc., Beijing, China), sCD40L (100 ng/mL; Sino Biological Inc., Beijing, China), or IFN-γ combined with sCD40L for 48 hours. TAO-OFs and CD40-knockdown TAO-OFs were co-cultured with autologous T cells (OFs: T cells = 1: 10) for 48 hours or not. TAO-OFs were stimulated with either PBS, IFN-γ neutralizing antibody (1 ug/mL; Proteintech, ORD, USA), autologous T cells, and IFN-γ Ab combined with and T cells for 48 hours. TAO-OFs were treated with either SB203580 (30 uM, p38 inhibitor), PD98059 (30 uM, ERK1/2 inhibitor), SP600125 (30 uM, JNK inhibitor), or PDTC (100 uM, NF-κB inhibitor) (all from MedChemExpress, NJ, USA) for 30 minutes, and then co-cultured with autologous T cells for 24 hours or not. Supernatant of cell culture from each experiment was collected and centrifuged at 1000 g for 10 minutes to remove debris. HA, IL-6, IL-8 (all from R&D Systems, Minneapolis, MN, USA), and sICAM-1 (4A Biotech, Beijing, China) were quantified in triplicate by ELISA according to the manufacturer’s protocols.