P mapk14

TC-71 and MHH-ES-1 cells were incubated with viscum, TT or viscumTT in increasing concentrations for 24 h. The cells were washed twice with phosphate-buffered saline and incubated in Lysis Buffer 17 (R&D systems, Minneapolis, MN) containing protease inhibitors (complete Protease Inhibitor Cocktail Tablets, Roche Diagnostics GmbH) to obtain cell lysates. Protein concentration was determined using Bradford solution (Bio-Rad, Munich, Germany). TC-71 and MHH-ES-1 cell lysates (30 μg protein/lane) were separated on SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad system) and blocked with 5% non-fat milk in 50 mM Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature. Blots were incubated overnight at 4 °C in TBST containing 5% BSA and primary antibody, washed thrice in TBST and incubated 1 h with HRP-conjugated secondary antibodies (anti-rabbit and anti-mouse, Bio-Rad) then visualized by ECL (Thermo Scientific) on a Molecular Imager ChemiDoc (Bio-Rad). Primary antibodies were directed against p-MAPK14 (Thr180/Tyr182, #9211 Cell Signaling Technology, Danvers, MA, USA), LC3B (#2775 Cell Signaling Technology), EIF2AK3 (#3192, Cell Signaling Technology), p-MAPK8 (sc-6254, Santa Cruz biotechnology, Dallas, TX, USA), HSPA5 (#G8918, Sigma-Aldrich) and ß-actin conjugated directly to peroxidase (#A3854, Sigma-Aldrich).

HCASMC cells were distributed in six-well plates at 5 × 10⁵/dish, and the corresponding treatment was carried out for 24 h. Then, cell lysis was conducted with RIPA lysis kit (Beyotime) including phenylmethanesulfonyl fluoride. Quantification of total protein was detected with BCA protein assay kit (Beyotime). Total proteins (20 μg) were separated using 7.5–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The resolved protein was subsequently transferred onto a nitrocellulose membrane. Membranes were blocked with quick blocking buffer (Beyotime) for 10 min at room temperature, and then incubated with primary antibodies at 4°C for 15 h, including p-STAT3, p-MAPK14, p-AKT (1:1,000; Cell Signaling Technology, Danvers, MA, USA), p-PI3K, PI3K, AKT, mTOR, and p-mTOR (1:1,000; Proteintech Group, Wuhan, China). Membranes were washed and then incubated in secondary antibodies (1:10,000) at room temperature for 1 h. Proteins were imaged by the Bio-Rad Chemi Doc XRS imaging system (Bio-Rad, USA).

The tissue specimens were dewaxed and subsequently blocked for about 30 min with goat serum. Primary antibodies against antirabbit-GDF15 [1:2000], p-MAPK14 [1:1000], CD31 [1:1000], and p16 [1:1000] were bought from Cell Signaling Technology (Beverly, MA, USA) and incubated overnight at 4 °C on tissue specimens. Following that, the specimens were reacted for 20 min at 37 °C using the secondary antibody (goat antirabbit IgG, 1:1000 dilution). Following multiple rinsing with phosphate-buffered solution, the samples were reacted with diaminobenzidine. A yellow–brown color in the nuclei or cytosol portion of cells was regarded as positive expression of GDF15, p-MAPK14, p16, and CD31. The proportion of positively stained cells was calculated as follows: overall number of positive cells/total cell count × 100.