Fig. 7.

Celastrus orbiculatus stem extract (COE) regulates the protein interaction between polypyrimidine tract binding protein 1 (PTBP1) and alpha actinin-4 (ACTN4). (A) Western blotting was used to detect the expression of ACTN4 and PTBP1 in the COE and control proteins; protein degradation degree is expressed according to the band colour shade. (B, C) A hot melting curve drawn according to the protein expression levels. If the hot melting curve shifted to the right, the amount of undegraded protein in the COE group was increased compared with the control group. The melting curve of PTBP1 shows no rightward shift (B), whereas the melting curve of ACTN4 exhibits a rightward shift (C). (D) The graph shows the cell survival rate, with the Y-axis representing the cell viability (%) and the X-axis representing the concentration of COE. AGS short hairpin negative control (shNC) half maximal inhibitory concentration (IC₅₀) is 109.9 μg/mL and AGS shACTN4 IC₅₀ is >200 μg/mL. (E) The positive proximity ligation assay (PLA) signal is generated when the spatial distance between two target antigens (epitopes) is within 40 nm. Each PLA signal is composed of approximately 1000 bound fluorescent probes, which together form a clear red dot. (F) Immunofluorescence (IF) staining was performed to observe the expression of PTBP1 and ACTN4 in gastric cancer (GC) cells. Data are representative of three independent experiments. DAPI: 4′,6-diamidino-2-phenylindole.