Figure legends
Fig. 1. GGC alters STAT3 activation. (A) The structure of Ginkgolide C (GGC). (B) A549, H1299, and HEL 299 cells were treated with the indicated concentration of GGC for 24 h and viability was measured. (C) A549 cells were treated for indicated time intervals with GGC 30 µM and western blotting was executed. (D)A549 cells were treated as described above in panel (C) and STAT3 levels were determined by EMSA. (E) A549 cells were treated with GGC 30 µM for 12 h and STAT3 distribution was measured.(F) A549 cells were incubated with various concentrations of GGC and western blotting was executed. (G) Western blotting to analyze STAT5 (Tyr694/Tyr699) expression. (H) A549 cells were transfected with empty vector (pcDNA) or STAT3-C Flag pRc/CMV (300 ng) for 24 h. Whole-cell extracts were prepared and Western blot was executed. (I) A549 cells were transfected with Empty vector (pcDNA) or STAT3-C Flag pRc/CMV were treated with or without of GGC for 24 h and cell viability was analyzed.
Fig. 2. GGC blocks IL-6 promoted STAT3 activation and increases PTPε levels. (A) H1299 cells were treated with the indicated concentrations of GGC for 12 h and IL-6 (20 ng/ml) for 15 min. Thereafter western blot was executed. (B) H1299 cells were treated with GGC 30 µM and stimulated with IL-6 as described above. Western blot was executed on the cell lysates. (C) A549 cells were treated as described above in panel (B) and western blotting was executed. (D) H1299 cells were transfected with STAT3 and treated with GGC for 12 h. After stimulation with IL-6 (20 ng/ml) for 15 min, luciferase assay was conducted. (E) A549 cells were exposed to pervanadate and 30 µM of GGC for 12 h followed by western blotting. (F) A549 cells were treated with GGC for 12 h and western blot analysis was executed. (G) A549 cells were processed as elaborated above and RT-PCR was done. (H) A549 cells were transfected with PTPε-specific siRNA and scrambled RNA. After 24 h, the cells were exposed to GGC 30 µM for 12 h. Lysates from the cells were determined by Western blot. (I) A549 cells were transfected and treated with as described above in panel (H) and western blotting was executed.
Fig. 3. GGC promotes apoptosis and suppresses proliferation. (A) A549 cells were treated with GGC 30 µM for 24 h. and apoptosis was examined by Cell cycle Analysis, Annexin V, and TUNEL assays. H1299 cells were treated with GGC 30 µM. After 12 h, cells were exposed to IL-6 (20 ng/ml) for 12 h and apoptosis was determined (B) A549 cell were treated with GGC for 24 h. H1299 cells were exposed to GGC. After 12 h, IL-6 (20 ng/ml) was added in these cells for 12 h. Thereafter western blotting was executed. (C) The cells were seeded in 16-well E-plates and treated with various concentration of GGC and IL-6 as elaborated above and proliferation assay was performed.
Fig. 4. GGC attenuates levels of tumorigenic proteins. (A) A549 cells were treated with GGC for 24 h and western blotting was executed.(B) H1299 cells were exposed to GGC 30 µM. After 12 h, IL-6 (20 ng/ml) was added for 24 h and western notting was conducted.(C) A549 cells were treated with various concentration of GGC and RT-PCR was done for different genes. (D) H1299 cells were treated with GGC and IL-6 as elaborated above. (B) and RT-PCR was conducted. (E) A549 cell were transfected with PTPε-specific siRNA and scrambled RNA. After 48 h, the cells were treated with GGC 30 µM for 24 h and western blotting was executed. (F) A549 cells transfected with STAT3-C Flag pRc/CMV (300 ng) were treated with or without of GGC for 24 h and western blot analysis was conducted.
Fig. 5. Anti-tumor actions of GGC. (A) In vivo experimental design. (B) Necropsy photographs of mice carrying implanted tumors. (C) Tumor volume data. ** p < 0.01 compared the control. (D) Tumor weight was measured on Day 25. * p < 0.05 compared the control. (E) Body weight was calculated on the indicated days.
Fig. 6. GGC alters the levels of oncogenic markers in tissues. (A) GGC treated mice tissues were studied by immunohistochemical staining. Quantification data was represented as mean ± SD on right panel. (B-E) Western blot data for different markers in whole cell extracts from mouse tissues.