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.