Fig. 4.

CTK7A induced intrinsic apoptosis by mitochondrial translocation of Noxa in CoCl₂-treated GCCs. (A) Western blot (n = 3) showing status of Noxa in the mitochondria and cytosolic fraction of AGS cells treated with CoCl₂, 200 µM, or in combination with CTK7A (100 µM) for 24 h. COX IV and GAPDH were used as loading controls for the mitochondrial and cytosolic fractions, respectively. Bars depict Noxa expression normalized to COX IV in the mitochondrial fraction (mean ± SEM, n = 3), *P < 0.05. (B) Confocal microscopy of AGS cells (n = 3) treated with CoCl₂ (200 µM) and/or CTK7A (100 µM) for 24 h showed high Noxa translocation to mitochondria in CTK7A + CoCl₂-treated cells. Nuclei were stained with DAPI. Scale bar 10 µm. (C) Confocal microscopic image showing cytochrome c release from mitochondria in the above mentioned experimental groups. DAPI staining was for nucleus. Scale bar 10 µm. (D) Immunoblotting (n = 3) of whole cell lysates prepared from AGS cells after treatment with CoCl₂ (200 µM) or hypoxia (1% O₂) alone or in combination with CTK7A (100 µM) for 24 h indicated status of Noxa, cleaved caspase 3 and cleaved caspase 9. Error bars, mean ± SEM, n = 3,*P < 0.05. (E) AGS cells were treated with 200 µM CoCl₂ alone or in combination with CTK7A (100 µM) or left untreated. Cells were harvested and stained with Annexin V PE/7-AAD dyes. A representative dot plot (n = 4) indicated striking increase in apoptosis in CTK7A + CoCl₂-treated GCCs as compared to other treatment groups.% cell death were compared between various treatment groups (mean ± SEM, n = 4), *P < 0.05.