Effect of Bcl‐xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl‐x_L overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated Bcl‐x_L overexpression (EPO‐off‐Bcl‐x_L) was established using the Tet‐off system. The expression level of Bcl‐x_L in EPO‐off‐Bcl‐x_L cells was tightly regulated by doxycycline in a dose‐dependent manner. Bcl‐x_L overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl‐x_L overexpression suppressed apoptosis by inhibiting the activation of caspase‐3 and ‐7. Simultaneously, Bcl‐x_L overexpression also delayed autophagy, characterized by LC3‐II accumulation. Immunoprecipitation analysis with a Flag‐tagged Bcl‐x_L revealed that Bcl‐x_L interacts with Bax and Bak, essential mediators of caspase‐dependent apoptosis, as well as with Beclin‐1, an essential mediator of autophagy, and may inhibit their pro‐cell death function. Taken together, it was found that Bcl‐x_L overexpression inhibits both apoptosis and autophagy in rCHO cell culture. Biotechnol. Bioeng. 2009;103: 757–766. © 2009 Wiley Periodicals, Inc.     

Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed-batch culture: effect of nutrient supplementation

Upon nutrient depletion during recombinant Chinese hamster ovary (rCHO) cell batch culture, cells are subjected to apoptosis, type I programmed cell death (PCD), and autophagy which can be type II PCD or a cell survival mechanism. To investigate the effect of nutrient supplementation on the two PCDs and protein production in rCHO cells, an antibody-producing rCHO cell line was cultivated in batch and fed-batch modes. The feed medium containing glucose, amino acids, and vitamins was determined through flask culture tests and used in bioreactor cultures. In the bioreactor cultures, the nutrient feedings extended the culture longevity and enhanced antibody production. In addition, cells in the fed-batch culture showed delayed onset of both apoptosis and autophagy, compared with those in the batch culture. The inhibition of apoptosis was demonstrated by a decreased amount of cleaved caspase-7 protein and less fragmentation of chromosomal DNA. Concurrently, reduced LC3 conversion, from LC3-I to LC3-II, was observed in cells that received the feeds. Cultivation with pharmacological autophagy inducer (rapamycin) or inhibitor (bafilomycin A1) indicated that autophagy is necessary for the cells to survive under nutrient depletion. Taken together, the delayed and relieved cell death by nutrient supplementation could improve antibody production.     

Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture

During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310–610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase‐3, caspase‐7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumulation of LC3‐II, a widely used autophagic marker, which was determined by Western blot analysis and confocal microscopy. When glucose and glutamine concentrations were measured during the cultures, glucose and glutamine concentrations in the culture medium at various osmolalities (310–610 mOsm/kg) showed no significant differences. This result suggests that induction of PCD by hyperosmotic stress occurred independently of nutrient depletion. Taken together, autophagy as well as apoptosis was observed in rCHO cells subjected to hyperosmolality. Biotechnol. Bioeng. 2010;105: 1187–1192. © 2009 Wiley Periodicals, Inc.     

Effect of Bcl‐xL overexpression on sialylation of Fc‐fusion protein in recombinant Chinese hamster ovary cell cultures

The sialic acid of glycoproteins secreted by recombinant Chinese hamster ovary (rCHO) cells can be impaired by sialidase under culture conditions which promote the extracellular accumulation of this enzyme. To investigate the effect of Bcl‐x_L overexpression on the sialylation of glycoproteins produced in rCHO cell culture, two rCHO cell lines producing the same Fc‐fusion protein, which were derived from DUKX‐B11 and DG44, respectively, were engineered to have regulated Bcl‐x_L overexpression using the Tet‐off system. For both cell lines, Bcl‐x_L overexpression improved cell viability and extended culture longevity in batch cultures. As a result, a maximum Fc‐fusion protein titer increased by Bcl‐x_L overexpression though the extent of titer enhancement differed between the two cell lines. With Bcl‐x_L overexpression, the sialylation of Fc‐fusion protein, which was assessed by isoelectric focusing gel and sialic acid content analyses, decreased more slowly toward the end of batch cultures. This was because Bcl‐x_L overexpression delayed the extracellular accumulation of sialidase activity by reducing cell lysis during batch cultures. Taken together, Bcl‐x_L overexpression in rCHO cell culture increased Fc‐fusion protein production and also reduced the impairment of sialylation of Fc‐fusion protein by maintaining high viability during batch cultures. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1133–1136, 2015     

Effect of sodium butyrate on autophagy and apoptosis in Chinese hamster ovary cells

Sodium butyrate (NaBu), which is widely used in recombinant Chinese hamster ovary cell (rCHO) cultures for high-level expression of therapeutic proteins, is known to induce apoptosis in a dose-dependent manner. Lately, the significance of autophagy has increased in the field of CHO cell culture due to the fact that autophagy is related to the programmed cell death mechanism. To determine the effect of NaBu on autophagy as well as apoptosis of rCHO cells, rCHO cells producing erythropoietin were subjected to NaBu treatment. NaBu treatment up to 5 mM increased cleaved forms of PARP, caspase-3, and Annexin V positive population, confirming the previous results that NaBu induces apoptosis. Concurrently, NaBu treatment increased the level of accumulation of the autophagic marker, LC3-II, independently of nutrient depletion, suggesting that NaBu induces autophagy. To elucidate the potential role of autophagy induced by NaBu, a representative autophagy inducer (rapamycin) or an inhibitor (bafilomycin A1) was added to cultures together with NaBu. It was found that autophagy had the potential role of a positive cell survival mechanism under NaBu treatment. Furthermore, gradual reduction in mitochondrial membrane potential/mass and recruitment of a mitophagy protein, Parkin, to the mitochondria were observed under NaBu treatment, suggesting that this positive function of autophagy might be mediated by the autophagic removal of damaged mitochondria. Taken together, autophagy was observed in rCHO cell culture under NaBu treatments and the results obtained here support the positive effects of autophagy induced by NaBu treatments.     

Bcl‐xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33°C and 37°C

Overexpression of bcl‐xL in recombinant Chinese hamster ovary (rCHO) cells has been known to suppress apoptotic cell death and thereby extend culture longevity during batch culture. However, its effect on specific productivity (q) of rCHO cells is controversial. This study attempts to investigate the effect of bcl‐xL overexpression on q of rCHO cells producing erythropoietin (EPO). To regulate the bcl‐xL expression level, the Tet‐off system was introduced in rCHO cells producing EPO (EPO‐off‐bcl‐xL). The bcl‐xL expression level was tightly controlled by doxycycline concentration. To evaluate the effect of bcl‐xL overexpression on specific EPO productivity (q_EPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The q_EPO at 33°C and 37°C in the presence of 100 ng/mL doxycycline (without bcl‐xL overexpression) were 4.89 ± 0.21 and 3.18 ± 0.06 μg/10⁶cells/day, respectively. In the absence of doxycycline, bcl‐xL overexpression did not affect q_EPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the q_EPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl‐xL overexpression

Bcl‐x_L, a member of the Bcl‐2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl‐x_L overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐x_L expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) and MS analysis. The consequences of Bcl‐x_L overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐x_L overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif‐containing GTPase‐activating protein 1), cell proliferation (dihydrolipoamide‐S‐acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl‐x_L not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects. Biotechnol. Bioeng. 2010; 105: 358–367. © 2009 Wiley Periodicals, Inc.     

Biophysical changes reduce energetic demand in growth factor–deprived lymphocytes

Cytokine regulation of lymphocyte growth and proliferation is essential for matching nutrient consumption with cell state. Here, we examine how cellular biophysical changes that occur immediately after growth factor depletion promote adaptation to reduced nutrient uptake. After growth factor withdrawal, nutrient uptake decreases, leading to apoptosis. Bcl-x_L expression prevents cell death, with autophagy facilitating long-term cell survival. However, autophagy induction is slow relative to the reduction of nutrient uptake, suggesting that cells must engage additional adaptive mechanisms to respond initially to growth factor depletion. We describe an acute biophysical response to growth factor withdrawal, characterized by a simultaneous decrease in cell volume and increase in cell density, which occurs before autophagy initiation and is observed in both FL5.12 Bcl-x_L cells depleted of IL-3 and primary CD8⁺ T cells depleted of IL-2 that are differentiating toward memory cells. The response reduces cell surface area to minimize energy expenditure while conserving biomass, suggesting that the biophysical properties of cells can be regulated to promote survival under conditions of nutrient stress.     

Disruption of the VDAC2–Bak interaction by Bcl-xS mediates efficient induction of apoptosis in melanoma cells

The proapoptotic B-cell lymphoma (Bcl)-2 protein Bcl-x_S encloses the Bcl-2 homology (BH) domains BH3 and BH4 and triggers apoptosis via the multidomain protein Bak, however, the mechanism remained elusive. For investigating Bcl-x_S efficacy and pathways, an adenoviral vector was constructed with its cDNA under tetracycline-off control. Bcl-x_S overexpression resulted in efficient apoptosis induction and caspase activation in melanoma cells. Indicative of mitochondrial apoptosis pathways, Bcl-x_S translocated to the mitochondria, disrupted the mitochondrial membrane potential and induced release of cytochrome c, apoptosis-inducing factor and second mitochondria-derived activator of caspases. In melanoma cells, Bcl-x_S resulted in significant Bak activation, and Bak knockdown as well as Bcl-x_L overexpression abrogated Bcl-x_S-induced apoptosis, whereas Mcl-1 (myeloid cell leukemia-1) knockdown resulted in a sensitization. With regard to the particular role of voltage-dependent anion channel 2 (VDAC2) for inhibition of Bak, we identified here a notable interaction between Bcl-x_S and VDAC2 in melanoma cells, which was proven in reciprocal coimmunoprecipitation analyses. On the other hand, Bcl-x_S showed no direct interaction with Bak, and its binding to VDAC2 appeared as also independent of Bak expression. Suggesting a new proapoptotic mechanism, Bcl-x_S overexpression resulted in disruption of the VDAC2–Bak interaction leading to release of Bak. Further supporting this pathway, overexpression of VDAC2 strongly decreased apoptosis by Bcl-x_S. New proapoptotic pathways are of principle interest for overcoming apoptosis deficiency of melanoma cells.     

Effect of Akt overexpression on programmed cell death in antibody-producing Chinese hamster ovary cells

Upon nutrient deprivation, Chinese hamster ovary (CHO) cells are subjected to two types of programmed cell death, apoptosis and autophagy. CHO cell engineering, as a means to improve foreign protein production, has focused mainly on anti-apoptosis. In this study, to determine the effect of Akt, which is known to regulate both apoptosis and autophagy, on cell survival and foreign protein production, constitutively active Akt was overexpressed in antibody-producing cells. Compared with the control cells, Akt overexpressing cells showed delayed onset of apoptosis and autophagy during batch culture. The inhibition of apoptosis was demonstrated by reduced amount of cleaved poly(ADP-ribose) polymerase and caspase 3 proteins and less fragmentation of chromosomal DNA. Moreover, under nutrient-limiting conditions, decreased level of autophagosome accumulation was observed in Akt overexpressing cells by the less accumulation of the 16kDa form of LC3-II and autophagic vacuoles. Taken together, the overexpression of constitutively active Akt in CHO cells could delay the onset of both types of programmed cell death during batch culture.     

Denitrosylation of S-nitrosylated OGT is triggered in LPS-stimulated innate immune response

Rad is a member of a subclass of small GTP-binding proteins, the RGK family. In the present study we investigated the role of Rad protein in regulating cardiomyocyte viability. DNA fragmentation and TUNEL assays demonstrated that Rad promoted rat neonatal cardiomyocyte apoptosis. Rad silencing fully blocked serum deprivation induced apoptosis, indicating Rad is necessary for trigger cardiomyocyte apoptosis. Rad overexpression caused a dramatic decrease of the anti-apoptotic molecule Bcl-x_L, whereas Bcl-x_L overexpression protected cardiomyocytes against Rad-induced apoptosis. Rad-triggered apoptosis was mediated by the activation of p38 MAPK. The p38 blocker SB203580 effectively protected cardiomyocytes against Rad-evoked apoptosis.     

Effects of dissolved oxygen levels and the role of extra- and intracellular amino acid concentrations upon the metabolism of mammalian cell lines during batch and continuous cultures

The effects of dissolved oxygen and the concentration of essential amino acids upon the metabolism of two mammalian cell lines (rCHO producing human active (t-PA) and a mouse-mouse hybridoma) were investigated in batch, chemostat, and perfusion cultures. Intracellular amino acid concentrations were measured for both cell lines during repeated batch cultures and the K_S-values for the essential amino acids were calculated using Monod equations via computer simulation. The K_S-values were in the range of 10 mmol L⁻¹ and the pool of most intracellular amino acids remained constant at about 10–100 fold higher in concentration than in the medium. No significant differences were observed between the hybridoma and CHO cell. The specific nutrient uptake rates corresponded with the cell specific growth rate and the effects of reduced dissolved oxygen concentrations only became evident when the DO dropped below 5% of air saturation (critical concentration below 1%). Nevertheless, a correlation between nutrient concentration and specific oxygen uptake was detected. This revised version was published online in June 2006 with corrections to the Cover Date.     

Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro

Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of spinal cord motoneurons (MNs), resulting in muscular atrophy and weakness. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and decreased SMN protein. SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoproteins and pre-mRNA splicing requirements. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to SMN degeneration in SMA are still unknown. Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in neurodegenerative diseases remain largely unknown. In particular, it is unclear whether autophagosome accumulation is protective or destructive, but the accumulation of autophagosomes in the neuritic beadings observed in several neurite degeneration models suggests a close relationship between the autophagic process and neurite collapse. In the present work, we describe an increase in the levels of the autophagy markers including autophagosomes, Beclin1 and light chain (LC)3-II proteins in cultured mouse spinal cord MNs from two SMA cellular models, suggesting an upregulation of the autophagy process in Smn (murine survival motor neuron protein)-reduced MNs. Overexpression of Bcl-x_L counteracts LC3-II increase, contributing to the hypothesis that the protective role of Bcl-x_L observed in some SMA models may be mediated by its role in autophagy inhibition. Our in vitro experimental data indicate an upregulation in the autophagy process and autophagosome accumulation in the pathogenesis of SMA, thus providing a valuable clue in understanding the mechanisms of axonal degeneration and a possible therapeutic target in the treatment of SMA.     

Effect of Bcl‐xL overexpression on lactate metabolism in chinese hamster ovary cells producing antibody

Previously, overexpression of anti‐apoptotic proteins, such as E1B‐19K and Aven, was reported to alter lactate metabolism of CHO cells in culture. To investigate the effect of Bcl‐x_L, a well‐known anti‐apoptotic protein, on lactate metabolism of recombinant CHO (rCHO) cells, two antibody‐producing rCHO cell lines with regulated Bcl‐x_L overexpression (CS13*‐0.02‐off‐Bcl‐x_L and CS13*‐1.00‐off‐Bcl‐x_L) were established using the Tet‐off system. When cells were cultivated without Bcl‐x_L overexpression, the specific lactate production rate (q_Lac) of CS13*‐0.02‐off‐Bcl‐x_L and CS13*‐1.00‐off‐Bcl‐x_L were 7.32 ± 0.37 and 6.78 ± 0.56 pmol/cell/day, respectively. Bcl‐x_L overexpression, in the absence of doxycycline, did not affect the q_Lac of either cell line, though it enhanced the viability during cultures. Furthermore, activities of the enzymes related to glucose and lactate metabolism, such as hexokinase, glucose‐6‐phosphate dehydrogenase, lactate dehydrogenases, and alanine aminotransferase, were not affected by Bcl‐x_L overexpression either. Taken together, Bcl‐x_L overexpression showed no significant effect on the lactate metabolism of rCHO cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1594–1598, 2013     

Differential Interactions Between Beclin 1 and Bcl-2 Family Members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-x_L, Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-x_L was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-x_L. Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-x_L. Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-x_L and Beclin 1.     

Rapamycin treatment inhibits CHO cell death in a serum‐free suspension culture by autophagy induction

Rapamycin, a specific mTOR inhibitor, has been used as a chemical activator in autophagy research both in vitro and in vivo. Recently, autophagy has received attention as an anti‐cell death engineering target in addition to apoptosis in the Chinese hamster ovary (CHO) cell engineering field. Here, the effect of rapamycin and the subsequent autophagy induction is investigated on two CHO cell lines, DG44 host and an antibody‐producing recombinant CHO (rCHO), in a serum‐free suspension culture. In both cell lines, the rapamycin treatment delayed the viability drop and apoptosis induction. In particular, the improved cell viability of the antibody‐producing rCHO cell line resulting from the rapamycin treatment led to a 21% increase in the maximum antibody concentration. From observations that a rapamycin derivative, everolimus, demonstrated similar positive effects in both cell lines, but not FK‐506, which forms the same complex as rapamycin, but does not inhibit mTOR, it was demonstrated that the positive effects of rapamycin appear to be mTOR‐dependent. In addition, the cultivation with rapamycin and/or an autophagy inhibitor, bafilomycin A1, indicated that the autophagy induction is related to the positive effects of rapamycin. The genetic perturbation of the autophagy pathway through the regulation of the expression level of Beclin‐1, an important autophagy regulator, resulted in a delayed autophagy induction and apoptosis inhibition in response to the rapamycin treatment in the DG44 host cell line. Taken together, the results obtained in this study imply a positive role for autophagy and predict the usefulness of pro‐autophagy engineering in CHO cell cultures. Biotechnol. Bioeng. 2012; 109: 3093–3102. © 2012 Wiley Periodicals, Inc.     

Monitoring of autophagy in Chinese hamster ovary cells using flow cytometry

Recently, autophagy, which is a degradative process, has drawn attention as an anti-cell death engineering target in addition to apoptosis in recombinant Chinese hamster ovary (rCHO) cell cultures for enhanced production of therapeutic proteins. Appropriate autophagy monitoring methods, that are suitable for long term CHO cell cultures, are necessary in order to investigate the culture conditions that affect the autophagy pathway and to select appropriate engineering targets for autophagy control. Herein, detailed protocols for autophagy monitoring methods based on flow cytometry are provided using the GFP-LC3-overexpressing CHO DG44 host cell line or MDC-like molecules in rCHO cells grown as an adherent culture with serum-containing medium or suspension culture with serum-free medium. Furthermore, combined with the apoptosis detection based on the Annexin V-PS interaction, the simultaneous detection of autophagy and apoptosis is also described. It is anticipated that the protocols described herein will assist in the fast, high throughput monitoring of autophagy that can support other existing autophagy assays.     

Beyond Cell Death – Antiapoptotic Bcl-2 Proteins Regulate Migration and Invasion of Colorectal Cancer Cells In Vitro

Migration and invasion of malignant cells are prerequisites for cancer progression and metastasis. The Bcl-2 family of proteins consists of about 25 members and has been extensively studied in the context of apoptosis. Despite the fact that small molecules targeting Bcl-2 proteins have already entered clinical trials, very few studies investigated a role of antiapoptotic Bcl-2 proteins beside cell death in the context of metastasis. The aim of this study was to dissect a potential role of the antiapoptotic Bcl-2 proteins Mcl-1, Bcl-2 and Bcl-x_L on migration and invasion of colorectal cancer cells independent of their cell death control function. We used migration and invasion assays as well as three dimensional cell cultures to analyze colorectal cancer cell lines (HT29 and SW480) after siRNA mediated knockdown or overexpression of Mcl-1, Bcl-2 or Bcl-x_L. We observed neither spontaneous cell death induction nor impaired proliferation of cells lacking Mcl-1, Bcl-2 or Bcl-x_L. In contrast, knockdown of Mcl-1 led to increased proliferation. Strikingly, we demonstrate a profound impairment of both, migration and invasion, of colorectal cancer cells after Mcl-1, Bcl-2 or Bcl-x_L knockdown. This phenotype was completely revised in cells overexpressing Mcl-1, Bcl-2 or Bcl-x_L. The most pronounced effect among the investigated proteins was observed for Bcl-2. The data presented indicate a pivotal role of Mcl-1, Bcl-2 and Bcl-x_L for migration and invasion of colorectal cancer cells independent of their known antiapoptotic effects. Thus, our study illustrates novel antitumoral mechanisms of Bcl-2 protein targeting.     

BimL displacing Bcl-xL promotes Bax translocation during TNFα-induced apoptosis

Bcl-2 family proteins are implicated as essential regulators in tumor necrosis factor-α (TNFα)-induced apoptosis. Bim_L, a BH3-only member of Bcl-2 family, can directly or indirectly activate the proapoptotic Bax and the subsequent mitochondrial apoptotic pathway. However, the molecular mechanism of Bim_L activating Bax activation during TNFα-induced apoptosis is not fully understood. In this study, the role of Bim_L in Bax activation during TNFα-induced apoptosis was investigated in differentiated PC12 and MCF7 cells, with real-time single-cell analysis. The experimental results show that Bax translocated to mitochondria and cytochrome c (Cyt c) released from mitochondria after TNFα treatment. Furthermore, SP600125 (specific inhibitor of JNK) could inhibit the Cyt c release from mitochondria. Co-immunoprecipitation results show that, the interaction between Bcl-x_L and Bax decreased after TNFα treatment, while that between Bcl-x_L and Bim_L increased. Bax did not co-immunoprecipitate with Bim_L before or after the TNFα treatment. In addition, the increased interaction between Bim_L and Bcl-x_L was dynamically monitored by using fluorescence resonance energy transfer (FRET) technique. Most importantly, there was no evidence of Bim_L redistribution to mitochondria until cell apoptosis. By comprehensively analyzing these data, it is concluded that Bim_L displaces Bcl-x_L in the mitochondria and promotes Bax translocation during TNFα-induced apoptosis.