Fig. 3: CPX induces proliferation inhibition and autophagic death by promoting STAT3 ubiquitination in GC cells.

A Western blotting analysis of p-STAT3 (Ser727), p-STAT3 (Tyr705). STAT3, STAT1, and STAT6 in GC cells treated with a serial dose of CPX for 24 h. B RT-qPCR analysis of STAT3 mRNA levels in GC cells treated with a different dose of CPX for 24 h. Data were shown as mean ± SD (n = 3, n.s., not significant by unpaired Student’s t test). C Ubiquitination of STAT3 in GC cells treated with a serial dose of CPX for 24 h was detected using immunoprecipitation. D Western blotting analysis demonstrating STAT3 overexpression in the indicated GC cells with or without CPX for 24 h. E, F Colony formation of STAT3-overexpressing cell lines treated with CPX for 1–2 weeks. Cell colonies were stained with 0.1% crystal violet solution (E). Colony numbers were counted using ImageJ Plus software (F). Data were shown as mean ± SD (n = 3, *P < 0.05, ***P < 0.001, ****P < 0.0001). G, H Cells treated with CPX (MGC: 10 µM; AGS: 10 µM; SGC: 40 µM) for 24 h and then stained with annexin V-FITC/PI were determined using flow cytometry (G). The cell death rate was plotted followed by statistical analysis (H). Data were shown as mean ± SD (n = 3, **P < 0.01, *** P < 0.001, ****P < 0.0001).