Figure 3.

Exogenous GIMAP6 displays anti-apoptotic effects in Huh-7 stable transfectant cells. A, the GIMAP6 expression level in Huh-7 derivative cells was assessed by immunoblot analysis. After transfection with a recombinant pcDNA3.1(+) vector carrying the GIMAP6 sequence, stably expressing single colonies were picked under G418 selection (500 μg/ml) for 2 weeks. Three drug-resistant clones (Neg, TF1, and TF2) were analyzed for GIMAP6 expression, and these clones were found to show substantial variation in protein levels by immunoblot analysis. B and C, the effect of GIMAP6 on OA-mediated cell apoptosis. B, cells were treated with OA for 24 h, and the level of immunofluorescence staining was analyzed. The cleaved form of caspase-3 (Cell Signaling Technology, 9661S) was used as an apoptotic marker (green), and DAPI (blue) was used as a counterstain as well as a total cell dye. C, the GIMAP6-negative cell line (Neg) was found to have a relatively higher proportion of caspase-3-positive cells compared with the GIMAP6-expressing cell lines (TF1 and TF2). The data represent the average of two independent experiments (mean ± S.D.). ***, p < 0.001, Student's t test. D, GIMAP6 knockdown restored the apoptosis-prone phenotype of the stably expressing cells. The stably expressing cell line (TF2) was transfected with anti-GIMAP6 shRNA to knock down the exogenously expressed GIMAP6. Single colonies (2K1, 2K2, and 2K3) were picked after puromycin selection (2 μg/ml) for 1 week. The result showed that the OA effect on low-GIMAP6-expressing cells (2K2 and 2K3) was restored to the original apoptosis-prone status; this should be compared with the situation when high-GIMAP6-expressing cells (TF2 and 2K1) were subjected to the same treatment procedure. C3F is an antibody that recognizes both the full-length and cleaved form of caspase-3 (GeneTex, GTX110543). The caspase-3 cleavage percentage (Ratio %) was calculated as cleaved/(cleaved + full-length) caspase-3.