TUNEL assay Apoptotic DNA fragmentation in
TUNEL assay. Apoptotic DNA fragmentation in TMPyP4 tosylate was measured using a commercially available TUNEL assay kit (Thermo Fisher). Frozen cells were fixed with 10% paraformaldehyde/PBS. Apoptosis was determined by staining the 3′-OH ends of fragmented DNA with biotin-dNTP using DNA I klenow fragment at 37 °C for 2 h, followed by incubation with horseradish peroxidase-conjugated streptavidin and incubation with 3,3′-diaminobenzidine and H2O2. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence images were detected using a fluorescence microscope (Nikon Eclipse 80i). For quantitative analysis, at least 500 cells were counted for each sample.
Cell viability. Cell viability was quantified using the MTT colorimetric assay kit (Promega) as previously described . Briefly, cells were seeded in 48-well plates at a concentration of 5 × 103 cells/well for 24 h following treatment with rAd-AIFM1. The medium was replaced with 250 μl of fresh serum-containing medium per well and 25 μl of MTT dissolved in PBS per well, followed by incubation of the plates at 37 °C for 4 h. Next, 250 μl of an isopropanol/0.04 N HCl solution was adding into the wells, followed by incubation at room temperature for 15 min under constant shaking. Absorbance measurement (570 nm: test wavelength; 690 nm: reference wavelength) was used to detect the cell viability.
Luciferase reporter assay. The detailed methods have been described previously . Briefly, Hep3B cells were transfected with pGL3-caspase3/DRAM promoter reporter constructs for 12 h, followed by infection with rAd-AIFM1 for 24–48 h. Relative light unit values were normalized to the β-galactosidase signal. One microgram of each indicated promoter-reporter plasmid and pRSV β-galactosidase plasmid was used for all transfections.
Statistical analysis. All the data shown are the results of at least three independent experiments and are expressed as means ± SEM. The differences between groups were compared using Student's t-test. Differences were considered significant at confidence levels of p < 0.05, p < 0.01 and p < 0.001, as indicated.
Discussion Previous studies have demonstrated that full-length AIFM1 (∼67) is only a precursor, and it needs to be processed to a mature form (∼62 kDa) and a truncated form (∼57 kDa) by proteolytic cleavage to maintain mitochondrial respiratory complex I and induce apoptosis, respectively [6,7,11]. In this study, to our knowledge, we first identified the apoptosis-inducing ability of full-length AIFM1, although the exact mechanisms by which full-length AIFM1 induces apoptosis remain unclear. We believe that the special cell lines that we used might endow full-length AIFM1 with the ability to induce apoptosis. If true, rAd-AIFM1 encoding full-length AIFM1 might be an ideal HCC gene therapy. In the future, we will study whether full-length AIFM1 also induces apoptosis in other tumor cell lines, which will help us to deeply understand the function of full-length AIFM1 in inducing apoptosis. To our knowledge, full-length AIFM1 that can promote the transcription of caspase3 and DRAM has not been reported. AIFM1 has an NLS domain at its C-terminus, which enables the nuclear translocation of AIFM1 . Our unpublished data also demonstrated that the NLS domain is required for full-length AIFM1 to induce the transcription of caspase3 and DRAM. In the future, we will determine whether full-length AIFM1 is a transcriptional factor that promotes the transcription of caspase3 and DRAM or whether full-length AIFM1 only cooperates with other transcriptional factors to promote the transcription of caspase3 and DRAM. Generally, AIFM1 is regarded as a key molecule in the apoptosis signaling pathway . When apoptotic signals trigger the opening of mitochondrial MPTP, AIFM1 will induce the release of cytochrome c and apoptosis protease activating factor-1 (Apaf-1), both of which can activate the caspase cascade and then induce apoptosis . Moreover, cytoplasmic AIFM1 promotes the release of more AIFM1 from mitochondria, forming a self-amplifying loop that accelerates apoptosis . Here, our data further uncover an inhibitory effect of full-length AIFM1 on the proliferation of hepatoma cells that also contributes to the tumor inhibitory effect of full-length AIFM1. In the future, we will also detect whether full-length AIFM1 could suppress the proliferation of other tumor cells.