Hep3B cells obtained from the American Type Culture Collection were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (PS) at 37°C in a 5% CO₂ humid atmosphere. After 48 h of culture, the medium was removed from the Hep3B cells, which were then washed with PBS and incubated in the same fresh medium.

The parkin-YFP plasmid was provided by Dr J. Chung (Seoul National University, Seoul, Korea). To establish cell lines that stably expressed parkin-YFP, Hep3B cells were seeded into 6-well plates (2×10⁵ cells/well) for 24 h prior to transfection. The cells were transfected with 2 μg of parkin-YFP plasmid using Lipofectamine 2000 according to the manufacturer's instructions. Stably transfected Hep3B/parkin-YFP cells were selected by incubating the cells in medium containing 500 μg/ml of neomycin sulfate (G418) for 2 weeks. The over-expression of parkin-YFP was confirmed by observing cells under Zeiss LSM 700 laser-scanning confocal microscope (Goettingen, Germany).

Hep3B cells growing into 6-well plates (0.8×10⁵ cells/well) were transfected with siRNA. siRNAs against the human Mfn1, Opa1, Drp1 transcripts were purchased from Dharmacon (ON-TARGETplus SMARTpool siRNA) and used at a concentration of 10 nM. As a negative control, the same nucleotide was scrambled to generate a non-targeting siRNA. The siRNAs were transfected into Hep3B cells using Lipofectamine^® RNAiMAX per the manufacturer's instructions.

The reagents were obtained from commercial sources: DMEM and FBS were obtained from Gibco-BRL (Gaithersburg, MD, USA); rabbit polyclonal antibodies to human Mfn1 (sc-50330), Tom20 (sc-11415) and cyclin B1 (sc-752), and mouse monoclonal antibody to human CDK1 (sc-54) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal antibodies to human Drp1 (61112) and Opa1 (612606) were obtained from BD Transduction Laboratories (Lexington, KY, USA); rabbit polyclonal antibodies against human caspase-3 (#9662), caspase-7 (#9492) and p-Drp1 (Ser616) (#3455) as well as HRP-conjugated goat anti-rabbit and horse anti-mouse IgG antibodies were obtained from Cell Signaling Technologies (Danvers, MA, USA), as was also the RIPA buffer. Texas Red-conjugated goat anti-rabbit IgG antibody was obtained from Vector (Burlingame, CA, USA); mouse monoclonal antibodies against human β-actin (A5441) and α-tubulin (T5168) as well as Hoechst 33342, protein-A agarose, N-acetyl-L-cysteine (NAC), propidium iodide (PI), carbonyl cyanide m-chlorophenylhydrazone (CCCP) and the Annexin V-FITC apoptosis detection kit were obtained from Sigma-Aldrich (Irvine, CA, USA). G418 was purchased from Duchefa (Haarlem, The Netherlands). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl benzimidazole carbocyanine iodide (JC-1) and MitoSOX™ Red reagent were purchased from Molecular Probes (Eugene, OR, USA). Lipofectamine^® RNAiMAX reagent and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA, USA). The SuperSignal West Pico enhanced chemiluminescence western blotting detection reagent was purchased from Pierce (Rockford, IL, USA) and RNase A was purchased from Biosesang (Biosesang, Inc., Korea).

G-TTP was synthesized by LegoChem Biosciences Inc. (Daejeon, Korea) as previously described (18).

The cell viability was assessed using the Vi-Cell cell counter (Beckman Coulter, Miami, FL, USA) to perform an automated trypan blue exclusion assay.

The cells were washed twice with ice-cold PBS, resuspended in RIPA buffer and incubated at 4°C for 30 min. The lysates were centrifuged at 14,000 rpm for 20 min at 4°C. The protein concentrations of the cell lysates were determined with the Bradford protein assay reagent (Bio-Rad), and 30 μg of protein was loaded onto 7.5–15% SDS/PAGE gels. The proteins were transferred to nitrocellu-lose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The bands were visualized by incubation with 1:1,000 dilutions of primary antibody overnight at 4°C, followed by incubation with 1:2,000 dilutions of secondary antibody at room temperature for 1 h. The antibody signal was developed using the SuperSignal West Pico-enhanced chemiluminescence substrate and detected with a LAS-4000PLUS (Fuji Photo Film, Tokyo, Japan).

The cells (3×10⁷ cells) were washed in ice-cold Tris-based Mg²⁺/Ca²⁺-free buffer (135 mM NaCl, 5 mM KCl and 25 mM Tris-HCl pH 7.4). The mitochondrial and cytosolic fractions were isolated using the Mitochondrial Fractionation kit (Active Motif, Carlsbad, CA, USA). The cells were resuspended in cytosolic buffer and incubated on ice for 15 min. The cells were then homogenized on ice with a homogenizer operated at 60 strokes. The lysates were centrifuged at 3,000 rpm and 4°C for 15 min. The supernatant contained the cytosol, including the mitochondria. The supernatant was transferred to a microcentrifuge tube and centrifuged at 13,000 rpm and 4°C for 30 min to pellet the mitochondria. The mitochondrial pellets were lysed with mitochondrial buffer on ice for 15 min and then centrifuged at 13,000 rpm for 30 min at 4°C.

After being incubated with antibodies, the cell extracts were precipitated with protein A-Sepharose beads for 3 h and washed 3 times with an extraction buffer prior to boiling them in the SDS sample buffer. The immunoprecipitated proteins were separated using SDS-PAGE, and a western blot analysis was performed as described above.

The cells were cultured on coverslips and fixed with 4% paraformaldehyde for 1 h. The cells were then permeabilized with 0.2% Triton X-100 for 15 min and incubated with Tom20 antibody for 1 h at room temperature (RT). They were washed 3 times with PBS for 5 min each, incubated with a Texas Red-conjugated secondary antibody for 1 h at room temperature and counterstained with Hoechst 33342. A Zeiss LSM 700 laser-scanning confocal microscope at a magnification of ×40 (0.55 numerical aperture) was used to obtain and analyze the fluorescent images. The cells were divided into the following 3 groups based on their mitochondrial morphology: fragmented, cells that primarily contained mitochondria shorter than 2 μm; tubular and donuts, cells that primarily contained mitochondria between ~2 and 5 μm long; and elongated, cells that primarily contained mitochondria >5 μm. Three independent experiments were conducted, and 100 cells were scored per experiment.

Ice-cold 95% ethanol containing 0.5% Tween-20 was added to the cell suspension to a final concentration of 70% ethanol. The fixed cells were pelleted and washed in 1% BSA-PBS solution. They were re-suspended in 1 ml of PBS containing 11 Kunitz U/ml RNase A, incubated at 37°C for 1 h, washed once with BSA-PBS, re-suspended in PI solution (10 μg/ml), and incubated in the dark at 4°C for 30 min. The cells were then washed with PBS, and the DNA content was measured on an Epics XL (Beckman Coulter). The data were analyzed using the MultiCycle software, which allowed the simultaneous estimation of cell cycle parameters and apoptosis.

To measure the MMP, the cells were trypsinized, collected, stained with JC-1 and subjected to flow cytometry using an Epics XL flow cytometer (Beckman Coulter). The data were acquired and analyzed using the EXPO32 ADC XL 4 color software program.

The cells were trypsinized, collected and stained with the Annexin V-FITC apoptosis detection kit according to the manufacturer's instructions. After centrifugation, cells were resuspended in PBS and analyzed with Epics XL.

The cells were trypsinized, collected and stained with 5 μM MitoSox for 30 min at 37°C. After centrifugation, the cells were resuspended in PBS and analyzed with Epics XL.

At least three independent experiments were carried out in vitro. The results are expressed as the means ± SD from three experiments. The significance of differences was determined using the paired Kruskal-Wallis non-parametric test. A p-value <0.05 was considered significant.

Treating Hep3B cells with 1–80 μM G-TPP for 48 h significantly reduced their viability in a dose-dependent manner (Fig. 1A). Because the viability of Hep3B cells treated with 40 μM G-TTP for 48 h was ~50%, this concentration was used for further studies. G-TPP treatment reduced the viability of Hep3B cells in a time-dependent manner (Fig. 1B). To examine whether the reduced viability of G-TTP-treated Hep3B cells was due to apoptotic cell death, we performed various apoptosis assays. The caspase cleavage and mitochondrial membrane potential assays indicated that G-TPP at least partly induced Hep3B cell death via apoptosis (Fig. 1C and D). Notably, G-TPP treatment significantly increased the population of Hep3B cells that contained elongated mitochondria (Fig. 1E and F).

We examined whether the G-TPP-induced mitochondrial elongation was mediated by alterations in the mitochondrial fusion-regulating proteins Mfn1 and Opa1. The western blot assay showed that G-TPP did not increase the expression levels of Mfn1 and Opa1 (Fig. 2A). We then examined the effect of Mfn1 or Opa1 depletion on G-TPP-induced cell death. A viability assay showed that neither siMfn1 nor siOpa1 significantly alter G-TPP-induced cell death (Fig. 2B). As predicted, both siMfn1 and siOpa1 significantly increased the population of G-TPP-untreated Hep3B cells that contained fragmented mitochondria. However, treatment with G-TPP significantly increased the populations of both Mfn1- and Opa1-depleted Hep3B cells that contained elongated mitochondria (Fig. 2C and D). These data indicate that G-TPP-induced mitochondrial elongation is not mediated by an increase in the mitochondrial fusion proteins Mfn1 and Opa1. We next examined whether the G-TPP-induced mitochondrial elongation was mediated by alterations in the fission-regulating protein Drp1. The western blot assay showed that treatment with G-TPP markedly decreased the Drp1 level in Hep3B cells, particularly in the mitochondria (Fig. 3A). We next examined the effect of Drp1 depletion on G-TPP-induced cell death. siDrp1 did not significantly alter the viability of Hep3B cells treated with G-TPP (Fig. 3B). However, siDrp1 significantly increased the populations of both G-TPP-treated and untreated Hep3B cells that contained elongated mitochondria compared with scrambled siRNA (Fig. 3C). Confocal microscopy demonstrated that mitochondria are more aggregated in Hep3B cells treated with G-TPP plus siDrp1 than cells treated with G-TPP plus scrambled siRNA (Fig. 3D). These data indicated that G-TPP-induced mitochondrial elongation was caused by Drp1 reduction.

We investigated whether G-TPP suppressed the translocation of Drp1 into mitochondria in parkin-overexpressing Hep3B cells. We observed that parkin translocated into the mitochondria of parkin-overexpressing Hep3B cells, irrespective of G-TPP treatment. Additionally, parkin translocated into the fragmented mitochondria of parkin-overexpressing Hep3B cells treated with the representative mitophagy inducer CCCP (Fig. 4A). A western blot assay demonstrated that G-TPP decreased the expression level of Drp1 not only in Hep3B cells but also in parkin-overexpressing Hep3B cells. G-TPP also inhibited the translocation of Drp1 into mitochondria in Hep3B cells. Importantly, CCCP increased the expression level of Drp1 and augmented the mitochondrial translocation of Drp1 in the parkin-overexpressing Hep3B cells. G-TPP inhibited the translocation of Drp1 into mitochondria in the parkin-overexpressing Hep3B cells (Fig. 4B). The population of cells that contained elongated or fragmented mitochondria did not differ between control Hep3B and parkin-overexpressing Hep3B cells treated with G-TPP (Fig. 4C). Furthermore, the overexpression of parkin did not affect the level of G-TPP-induced cell death (Fig. 4D). These data indicate that G-TPP inhibited the mitochondrial translocation of Drp1 even in the parkin-overexpressing Hep3B cells, which may impair parkin-mediated mitophagy.

Flow cytometry revealed an increase in the percentage of G2-M phase cells and a concomitant decrease in the percentage of the G1 phase cells (Fig. 1C), which indicated that G-TPP induced G2-M phase cell cycle arrest in Hep3B cells. Therefore, we examined the association between mitochondrial elongation and cell cycle progression in G-TPP-treated Hep3B cells. We examined the level of CDK1 and cyclin B1 with a western blotting, which showed that G-TPP markedly decreased the expression level of CDK1. G-TPP also markedly reduced the activating phosphorylation of Drp1 (Ser616) (Fig. 5A). Because the CDK1-cyclin B1 complex is known to be associated with the activation of Drp1 via the phosphorylation of Drp1 at Ser616 (pDrp1-Ser616) (20), we examined the effect of G-TPP on the formation of the CDK1-cyclin B1 complex. The co-immunoprecipitation results indicated that G-TPP reduced the level of CDK1-cyclin B1 complex formation (Fig. 5B). These data indicated that G-TPP reduced the interaction between CDK1 and cyclin B1 and thereby inhibited its Drp1 activation and mitochondrial localization, which induced mitochondrial elongation.

Next, we examined the involvement of ROS in G-TPP-induced cell death and mitochondrial elongation in Hep3B cells. We observed that G-TPP increased the ROS level in Hep3B cells, and the ROS scavenger NAC remarkably inhibited the level of ROS in the G-TPP-treated Hep3B cells (Fig. 6A). Importantly, NAC significantly suppressed G-TPP-induced cell death (Fig. 6B). Various apoptosis assays showed that NAC suppressed G-TPP-induced apoptosis (Fig. 6C and D). NAC also significantly decreased the population of Hep3B cells treated with G-TPP cells that contained elongated mitochondria (Fig. 6E and F). These data indicated that ROS mediates mitochondrial elongation and cell death in G-TPP-treated Hep3B cells. To this end, we examined whether G-TPP reduced the interaction of CDK1 with cyclin B1 and the phosphorylation of Drp1 (pDrp1-Ser616) via ROS. Noticeably, NAC recovered the expression levels of Drp1, CDK1 and p-Drp1 (Ser616) (Fig. 7A); suppressed the G-TPP-induced dissociation of CDK1 from cyclin B1 in Hep3B cells (Fig. 7B); and recovered the recruitment of Drp1 to mitochondria in G-TPP-treated Hep3B cells (Fig. 7C). Flow cytometry revealed that NAC suppressed G-TPP-induced G2-M arrest (Fig. 7D). These results indicate that ROS played a pivotal role in mitochondrial elongation, which is mediated by the reduced association of CDK1 with cyclin B1 and decreased Drp1 phosphorylation at Ser616 in G-TPP-treated Hep3B cells.