Jnk inhibitor sp600125

Dihydromyricetin (CAS No. 27200-12-0, Bellancom) was ordered from Beijing Universal Materials Co., Ltd. (Beijing, China), with purity >98%, as detected by high performance liquid chromatography. DHM was dissolved in 100% dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution and was stored at −20°C. DHM solutions used in cell cultures were freshly prepared daily and the final concentration of DMSO did not exceed 0.1% throughout the study.
Apoptotic cells were quantified using an Annexin V-FITC/PI cell apoptosis detection kit from Becton Dickinson and Company (Franklin Lakes, NJ, USA) and monitored using flow cytometry (FACSCalibur, BD, Franklin Lakes, NJ, USA).
JNK inhibitor SP600125, ERK inhibitor U0126, and Caspase-3 inhibitor Ac-DEVD-CHO were purchased from Beyotime (Shanghai, China) and dissolved in DMSO at concentrations of 20, 10 and 30 μM, respectively.
Antibodies for p-JNK/JNK, p-ERK/ERK, and GRASP65 were bought from Abcam (Cambridge, MA, USA), antibody for Actin from Beyotime (Shanghai, China), and antibodies for p-p38/p38MAPK, cleaved-caspase-3, Bcl-2, and Bax from Cell Signaling Technology (Danvers, MA, USA).

MCF-7 and SGC7901 cells were seeded into 96-well plates at a density of 5 × 10³ cells/well for 24 h. The cells were incubated with different concentrations of DTP (0, 0.35, 0.7, and 1.4 μM) for 24 h followed by photoirradiation with an energy density of 14 J/cm² for 14 min or 16 J/cm² for 17 min at a wavelength of 650 nm. The cells were also incubated in the presence of 12 μM P38 inhibitor SB203580 (Beyotime, China), 12 μM JNK inhibitor SP600125 (Beyotime, China), 12 μM ERK inhibitor FR180204 (Beyotime), or 10 mM ROS inhibitor N-acetyl-L-cysteine (NAC; Beyotime). Each experimental group included four wells per group. Eight hours later, the cells were treated with 10 µL of Cell Counting Kit-8 (CCK-8; Beyotime) for 2 h at 37°C. Absorbance was measured at 450 nm using a microplate reader. The cell survival rate was calculated using the following formula: Cell survival rate (%) = ([OD_experimental − OD_blank]/[OD_control − OD_blank]) × 100.

Poly(I:C) (InvivoGen, San Diego, CA, USA) was used for the positive control. All antibodies used for Western blot, such as primary antibodies (RIG-I, MDA-5, MAVS, IκBα, p65, p-p65, JNK, c-Jun, β-actin), HRP-conjugated anti-mouse secondary antibody and HRP-conjugated anti-rabbit secondary antibody were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Alexa Fluor®647 conjugated anti-mouse IgG (CST, Danvers, MA, USA) and Alexa Fluor®647 conjugated anti-rabbit IgG (Beyotime, Shanghai, China) were used for the fluorescence confocal microscopy. JNK inhibitor SP600125 was purchased from Beyotime, Shanghai, China and Protein Marker 26616 was purchased from Thermo Fisher Scientific, Waltham, MA, USA.