Visualization of Golgia apparatus as an intracellular calcium store by laser scanning confocal microscope

Using laser scanning confocal microscopy,we have found that the in cells loaded with fluo-3/AM,highest intracellular Ca^2 in the perinuclear region is associated with the Golgi apparatus.The spatiotemporal subcellular distribution of Ca^2 in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated.PDGF,which releases Ca^2 from intracellular stores by inositol(1,4,5)-trisphosphate pathway ,produced a biphasic transient rise in intracellular calcium.The initial rise was resulted from a direct release of calcium from the golgi apparatus.Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin,an inhibitor of the Ca^2 transport ATPase of intracellular calcium store,Permeablizing the plasma membrane by 10μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store.These results suggest that the Golgi apparatus plays a role in Ca^2 regulation in signal transduction.     

Contributions of intracellular compartments to calcium dynamics: implicating an acidic store

Many cells show a plateau of elevated cytosolic Ca(2+) after a long depolarization, suggesting delayed Ca(2+) release from intracellular compartments such as mitochondria and endoplasmic reticulum (ER). Mouse pancreatic beta-cells show a thapsigargin-sensitive plateau ('hump') of Ca(2+) after a 30 s depolarization but not after a 10 s depolarization. Surprisingly, this hump depends primarily on compartments other than the mitochondria or ER. It is reduced by only 22% upon blocking mitochondrial Na(+)-Ca(2+) exchange and by only 18% upon blocking ryanodine or IP(3) receptors together. Further, the time course of ER Ca(2+) measured by a targeted cameleon does not depend on the duration of depolarizations. Instead, the hump is reduced 35% by treatments with the dipeptide glycylphenylalanine beta-napthylamide, a tool often used to lyse lysosomes. We show that this dipeptide does not disturb ER functions, but it lyses acidic compartments and releases Ca(2+) into the cytosol. Moreover, it induces leaks in and possibly lyses insulin granules and stops mobilization of secretory granules to the readily releasable pool in beta-cells. We conclude that the dipeptide compromises dense-core secretory granules and that these granules comprise an acidic calcium store in beta-cells whose loading and/or release is sensitive to thapsigargin and which releases Ca(2+) after cytosolic Ca(2+) elevation.     

An intracellular calcium store regulates protein synthesis in HeLa cells, but it is not the hormone-sensitive store.

There is considerable evidence, reviewed by Brostrom and Brostrom [1], that Ca2+ stores are involved in the regulation of protein synthesis. We provide evidence in HeLa cells that is consistent with their findings that depletion of Ca2+ stores and not changes in cytosolic free Ca2+ ([Ca2+]i) inhibit protein synthesis, but we also show that the mechanism leading to depletion is critical. Specifically, depletion of stores by the Ca(2+)-mobilizing hormone histamine does not inhibit protein synthesis. In assessing the role of Ca2+ stores in protein synthesis, experiments in certain cell types have been complicated by the use of Ca2+ ionophores, which simultaneously elevate [Ca2+]i and deplete Ca2+ stores. We have measured total cell Ca2+, [Ca2+]i and protein synthesis in HeLa cells under conditions that allowed evaluation of the separate contributions of stores and [Ca2+]i. Using 1,2-bis(2-aminophenoxyethane)-N,N,N'N'-tetraacetic acid (BAPTA) as an intracellular Ca2+, chelator and thapsigargin, which inhibits the membrane Ca(2+)-ATPase of storage vesicles, total cell Ca2+ can be depleted and this depletion is enhanced by extracellular EGTA which blocks Ca2+ influx; [Ca2+]i is actually lowered by BAPTA under these conditions. Protein synthesis is inhibited by BAPTA in the presence of EGTA and by thapsigargin with or without EGTA. However, histamine which with EGTA, affects an equal degree of Ca2+ depletion does not inhibit protein synthesis. Thus, it is suggested that Ca2+ stores are not homogeneous, and that the hormone-sensitive store specifically does not play a role in the regulation of protein synthesis. In this respect, the hormone-sensitive and insensitive stores do not functionally communicate and may be separately regulated.     

Vanadate stimulates rat pancreatic enzyme secretion through the release of calcium from an intracellular store

Orthovanadate accelerates 45Ca efflux and enzyme secretion from the rat pancreas incubated in either control (2.5 mM Ca) or nominally Ca-free buffers. Secretion induced by vanadate does not appear to be mediated by changes in either adenylate cyclase or sodium pump activity. Instead, vanadate appears to act at an intracellular site to cause the release of calcium from the same pool mobilised by acetylcholine. Vanadate action is not inhibited by DIDS. The effect of pH on vanadate action may be accounted for by changes in the distribution of the vanadates. Vanadyl sulphate inhibits secretion evoked by acetylcholine. This suggests that intracellular reduction of vanadate (+5 oxidation state) to the +4 oxidation state may account for an inhibitory component observed during stimulation with vanadate.     

Evidence that calcium acts as an intracellular messenger for adrenergic responses in human erythrocytes

Adrenergic stimulation of membrane protein phosphorylation has been studied in human erythrocytes. The adrenergic enhancement in phosphorylation of band 2 could be mimicked by the calcium-specific ionophore A23187 in the presence of 10 micron extracellular calcium. Experiments with the potassium ionophore, valinomycin, showed that potassium efflux was not the primary effector of the response. Trifluoperazine, an inhibitor of the Ca2+-dependent regulatory protein, calmodulin, inhibited phosphorylation stimulation by either norepinephrine or the calcium ionophore. The norepinephrine response was observed in the absence of extracellular calcium, implicating Ca2+ released from cellular bound pools in mediating the response.     

Evidence for receptor-mediated calcium entry and refilling of intracellular calcium stores in FRTL-5 rat thyroid cells.

The aim of the present study was to investigate the relationship between agonist-induced changes in intracellular free Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in Fura 2-loaded thyroid FRTL-5 cells. Stimulating the cells with ATP induced a dose-dependent increase in ([Ca2+]i). The ATP-induced increase in [Ca2+]i was dependent on both release of sequestered intracellular Ca2+ as well as influx of extracellular Ca2+. Addition of Ni2+ prior to ATP blunted the component of the ATP-induced increase in [Ca2+]i dependent on influx of Ca2+. In cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ induced a rapid increase in [Ca2+]i; this increase was inhibited by Ni2+. In addition, the ATP-induced influx of 45Ca2+ was blocked by Ni2+. Stimulating the cells with noradrenaline (NA) also induced release of sequestered Ca2+ and an influx of extracellular Ca2+. When cells were stimulated first with NA, a subsequent addition of ATP induced a blunted increase in [Ca2+]i. If the action of NA was terminated by addition of prazosin, and ATP was then added, the increase in [Ca2+]i was restored to control levels. Addition of Ni2+ prior to prazosin inhibited the restoration of the ATP response. In the presence of extracellular Mn2+, ATP stimulated quenching of Fura 2 fluorescence. The quenching was probably due to influx of Mn2+, as it was blocked by Ni2+. The results thus suggested that stimulating release of sequestered Ca2+ in FRTL-5 cells was followed by influx of extracellular Ca2+ and rapid refilling of intracellular Ca2+ stores.     

Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells. Evidence that an intracellular calcium pool and not an inositol phosphate regulates calcium fluxes at the plasma membrane

The depletion of an inositol 1, 4,5-trisphosphate-sensitive intracellular Ca2+ pool has been proposed to be the signal for Ca2+ entry in agonist-activated cells. Consistent with this idea, thapsigargin, which releases intracellular Ca2+ without inositol phosphate formation, has been reported to activate Ca2+ entry in certain cells. We now report the effects of thapsigargin on Ca2+ entry in parotid acinar cells. In fura-2-loaded parotid acinar cells, thapsigargin caused a sustained elevation of [Ca2+], but did not increase inositol phosphate formation. In the absence of extracellular Ca2+, the increase in [Ca2+], was transient, suggesting that thapsigargin activates both the release of Ca2+ from intracellular stores and the entry of Ca2+ from the extracellular space. In the absence of extracellular Ca2+, pretreatment with methacholine, an agonist believed to mobilize Ca2+ through the production of inositol 1,4,5-trisphosphate, inhibited but did not completely block the response to thapsigargin; likewise, pretreatment with thapsigargin inhibited the response to methacholine. In permeabilized cells, thapsigargin gradually released Ca2+, whereas inositol 1,4,5-trisphosphate caused a rapid and transient discharge of Ca2+. The simultaneous addition of thapsigargin with inositol 1,4,5-trisphosphate evoked a maximum Ca2+ release similar to that for inositol 1,4,5-trisphosphate alone, but the reuptake seen with inositol 1,4,5-trisphosphate alone was abolished. In intact cells, methacholine and thapsigargin together produced a greater initial release of Ca2+ than either alone, but they were not additive in the sustained phase of Ca2+ mobilization. These results demonstrate that the mechanisms for activation of Ca2+ entry by thapsigargin and methacholine are the same and are consistent with the idea that entry is initiated by the depletion of the intracellular inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. The results also indicate that, in contrast to previously proposed models, Ca2+ entry into agonist-activated cells occurs directly across the plasma membrane to the cytoplasm rather than through a cycle of uptake and release by the intracellular Ca2+ pool.     

Differential coding of humoral stimuli by timing and amplitude of intracellular calcium spike trains

The ubiquitous Ca2(+)-phosphoinositide pathway transduces extracellular signals to cellular effectors. Using a mathematical model, we simulated intracellular Ca2+ fluctuations in hepatocytes upon humoral stimulation. We estimated the information encoded about random humoral stimuli in these Ca2+ spike trains using an information-theoretic approach based on stimulus estimation methods. We demonstrate accurate transfer of information about random humoral signals with low temporal cutoff frequencies. In contrast, our results suggest that high-frequency stimuli are poorly transduced by the transmembrane machinery. We found that humoral signals are encoded in both the timing and amplitude of intracellular Ca2+ spikes. The information transmitted per spike is similar to that of sensory neuronal systems, in spite of several orders of magnitude difference in firing rate.     

Evidence for an intracellular niche for Bordetella pertussis in broncho-alveolar lavage cells of mice

Bordetella pertussis can attach, invade and survive intracellularly in human macrophages in vitro. To study the significance of this bacterial feature in vivo, we analyzed the presence of viable bacteria in broncho-alveolar lavage (BAL) cells of mice infected with B. pertussis. We found B. pertussis to be present in a viable state in BAL fluid cells until at least 19 days after infection, suggesting B. pertussis to be able to survive in those cells. This intracellular niche may play an important role in the pathogenesis of pertussis. Pertussis toxin and the RGD sequence of the virulence factor filamentous hemagglutinin (FHA) both play a role in the attachment of B. pertussis to human and mouse macrophages in vitro and we hypothesized these virulence factors to be required for invasion and subsequent intracellular survival of B. pertussis in macrophages in vivo. A B. pertussis double mutant, in which the FHA RGD motif was changed to RAD and the ptx genes were deleted, was also found in a viable state in BAL fluid cells, albeit at lower levels than the wild-type strain. In our model, uptake of B. pertussis by alveolar phagocytes in vivo is thus, at least in part, determined by the bacterial virulence factors FHA and pertussis toxin.     

Intracellular calcium uptake activated by GTP. Evidence for a possible guanine nucleotide-induced transmembrane conveyance of intracellular calcium

The GTP-activated Ca2+ release process we recently described (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464) was revealed in the preceding report to operate via a mechanism likely to be induced by close membrane association but which appears not to involve membrane fusion (Chueh, S. H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864). To determine more about the GTP-activated Ca2+ translocation process, effects of GTP on cells loaded with Ca-oxalate were investigated. Using permeabilized cells of both the N1E-115 neuroblastoma and DDT1MF-2 smooth muscle cell lines, 10 microM GTP activates a profound uptake of Ca2+ in the presence of oxalate, as opposed to release observed without oxalate. GTP stimulation of Ca2+ uptake was observed at oxalate concentrations (2 mM) only slightly augmenting Ca2+ uptake without GTP; with 8 mM oxalate (which alone induces linear Ca2+ accumulation) GTP still increases the rate of uptake. GTP-activated uptake in the presence of oxalate is completely reversed by 1 mM vanadate. 3% polyethylene glycol enhances the effect of GTP although GTP-activated uptake is still observed without polyethylene glycol. The Km for GTP for activation of Ca2+ uptake is 0.9 microM. Uptake is not activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp); however, GTP gamma S (but not GppNHp) completely blocks the action of GTP. GDP gives a delayed uptake response which is blocked by ADP, indicating its action arises from conversion to GTP. In the presence of ADP, GDP blocks the action of GTP; guanosine 5'-O-(2-thio)diphosphate, which does not activate uptake, also blocks the action of GTP. These data reveal almost exact correlation between parameters affecting GTP-activated uptake and release, strongly suggesting the same process mediates both events. To explain the opposite effects of GTP in the absence and presence of oxalate, it is proposed that GTP activates a transmembrane conveyance of Ca2+ between oxalate-permeable and -impermeable compartments.     

A distinct 2,5-di-(tert-butyl)-1,4-benzohydroquinone-sensitive calcium store in bovine adrenal chromaffin cells

The fluorescent Ca2+ indicator Fura 2 was used to monitor Ca2+ release induced by the Ins(1,4,5)P3-mobilizing agonist angiotensin II (Ag II), caffeine and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tuBHQ), in intact bovine adrenal chromaffin cells. Under low external Ca2+ conditions, tuBHQ, Ag II and caffeine elicited Ca2+ rises, indicating Ca2+ release from internal stores. Prior addition of Ag II had no noticeable effect on the extent of release of Ca2+ induced by tuBHQ. Stimulation of the cells with tuBHQ before either Ag II or caffeine, similarly had no effect on Ca2+ released by these two agonists. It was concluded, therefore, that there is a third intracellular Ca2+ store in bovine adrenal chromaffin cells, distinct and non-overlapping, from those sensitive to caffeine or Ins(1,4,5)P3-mobilizing agonists.     

Evidence that biosynthesis of platelet-activating factor (paf-acether) by human neutrophils occurs in an intracellular membrane

Human polymorphonuclear leukocytes (PMN) were incubated in the absence or presence of the calcium ionophore A23187 (6 microM) for 10 min at 37 degrees C. They were then lysed by nitrogen cavitation and fractionated using Percoll gradients. Three major fractions of increasing density corresponding to plasma membrane, intracellular membranes and secretory granules were detected using [3H]concanavalin A, NADH-dehydrogenase and beta-D-glucuronidase as respective markers. In both cases, the acetyltransferase activity responsible for biosynthesis of paf-acether (platelet-activating factor of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was detected in the intermediary fraction, the enzyme activity being increased 3-4-fold in stimulated cells. From the comparison with the distribution of various markers, it is concluded that in human PMN the final step of paf-acether assembly occurs in an intracellular membrane, possibly the endoplasmic reticulum.     

Evidence for multiple intracellular calcium pools in GH4C1 cells: investigations using thapsigargin.

The actions of thapsigargin (Tg), a plant sesquiterpene lactone, on Ca2+ homeostasis were investigated in digitonin-permeabilized GH4C1 rat pituitary cells. Tg (1 microM) caused a rapid and sustained increase in ambient Ca2+ concentration [( Ca2+]) and inhibited the rise in [Ca2+] induced by subsequent addition of TRH (100 nM), inositol 1,4,5-trisphosphate (IP3, 10 microM), or the nonhydrolyzable GTP analogue guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S, 10 microM). However, neither IP3 nor GTP gamma S pretreatment, which themselves release sequestered Ca2+, prevented the Ca2+ accumulation induced by Tg. Pretreatment with heparin (100 micrograms/ml, 10 min), an IP3 receptor antagonist, did not affect Ca2+ accumulation induced by Tg, although it abolished the rise in [Ca2+] induced by IP3. The ability of Tg to increase [Ca2+] was dependent on added ATP. We conclude that, in GH4C1 cells, Tg acts, in part, on TRH-, IP3- and GTP gamma S-sensitive Ca2+ pools; however, Tg also acts on an ATP-dependent pool of intracellular Ca2+ which is not sensitive to TRH, IP3 or GTP gamma S, indicating a complexity of intracellular Ca2+ pools not previously appreciated in these cells.     

Angiotensin II causes calcium entry into bovine adrenal chromaffin cells via pathway(s) activated by depletion of intracellular calcium stores

The characteristics and properties of the increase in cytosolic [Ca²⁺] that occurs in bovine adrenal medullary chromaffin cells on exposure to angiotensin II have been investigated. In fura-2 loaded cells exposure to a maximally effective concentration of angiotensin II (100 nM) caused a rapid, but transient increase in cytosolic [Ca²⁺] followed by a lower plateau that was sustained as long as external Ca²⁺ was present. In the absence of external Ca²⁺ only the initial brief transient was observed. In cells previously treated with thapsigargin in Ca²⁺-free medium to deplete the internal Ca²⁺ stores, angiotensin II caused no increase in cytosolic [Ca²⁺] when external Ca²⁺ was absent. Reintroduction of external Ca²⁺ to thapsigargin-treated, store-depleted cells caused a sustained increase in cytosolic [Ca²⁺] that was not further increased upon exposure to angiotensin II. Analysis of the data suggests that in bovine chromaffin cells angiotensin II causes Ca²⁺ entry via a pathway(s) activated as a consequence of internal store mobilization, and entry through this pathway(s) forms the majority of the sustained Ca²⁺ influx evoked by angiotensin II.     

Paradoxical buffering of calcium by calsequestrin demonstrated for the calcium store of skeletal muscle

Contractile activation in striated muscles requires a Ca²⁺ reservoir of large capacity inside the sarcoplasmic reticulum (SR), presumably the protein calsequestrin. The buffering power of calsequestrin in vitro has a paradoxical dependence on [Ca²⁺] that should be valuable for function. Here, we demonstrate that this dependence is present in living cells. Ca²⁺ signals elicited by membrane depolarization under voltage clamp were compared in single skeletal fibers of wild-type (WT) and double (d) Casq-null mice, which lack both calsequestrin isoforms. In nulls, Ca²⁺ release started normally, but the store depleted much more rapidly than in the WT. This deficit was reflected in the evolution of SR evacuability, E, which is directly proportional to SR Ca²⁺ permeability and inversely to its Ca²⁺ buffering power, B. In WT mice E starts low and increases progressively as the SR is depleted. In dCasq-nulls, E started high and decreased upon Ca²⁺ depletion. An elevated E in nulls is consistent with the decrease in B expected upon deletion of calsequestrin. The different value and time course of E in cells without calsequestrin indicate that the normal evolution of E reflects loss of B upon SR Ca²⁺ depletion. Decrement of B upon SR depletion was supported further. When SR calcium was reduced by exposure to low extracellular [Ca²⁺], release kinetics in the WT became similar to that in the dCasq-null. E became much higher, similar to that of null cells. These results indicate that calsequestrin not only stores Ca²⁺, but also varies its affinity in ways that progressively increase the ability of the store to deliver Ca²⁺ as it becomes depleted, a novel feedback mechanism of potentially valuable functional implications. The study revealed a surprisingly modest loss of Ca²⁺ storage capacity in null cells, which may reflect concurrent changes, rather than detract from the physiological importance of calsequestrin.     

Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells

L-type Ca²⁺ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba²⁺ influx in a depolarizing medium. In the absence of extracellular Ca²⁺, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba²⁺ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca²⁺-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in a Ca²⁺-free medium from 30 to 80 nM. This [Ca²⁺]_i increase declined slowly, giving rise to a modest elevation of [Ca²⁺]_i that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca²⁺-free medium. However, when the Ca²⁺ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca²⁺ channel activity, suggesting that a lower amplitude and prolonged release of Ca²⁺ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 μM) and ascomycin (5 μM), inhibitors of the Ca²⁺/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1 mM CaCl₂ to the Tg-treated cells increased [Ca²⁺]_i to 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca²⁺]_i increase elicited by passive depletion of internal Ca²⁺ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca²⁺]_i via store-dependent Ca²⁺ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca²⁺ channels in a myocyte model involves a balance between the differential Ca²⁺ sensitivities and opposing actions of PKC and calcineurin.     

Calcium isotope fractionation by intracellular amorphous calcium carbonate (ACC) forming cyanobacteria

The formation of intracellular amorphous calcium carbonate (ACC) by various cyanobacteria is a widespread biomineralization process, yet its mechanism and importance in past and modern environments remain to be fully comprehended. This study explores whether calcium (Ca) isotope fractionation, linked to ACC-forming cyanobacteria, can serve as a reliable tracer for detecting these microorganisms in modern and ancient settings. Accordingly, we measured stable Ca isotope fractionation during Ca uptake by the intracellular ACC-forming cyanobacterium Cyanothece sp. PCC 7425. Our results show that Cyanothece sp. PCC 7425 cells are enriched in lighter Ca isotopes relative to the solution. This finding is consistent with the kinetic isotope effects observed in the Ca isotope fractionation during biogenic carbonate formation by marine calcifying organisms. The Ca isotope composition of Cyanothece sp. PCC 7425 was accurately modeled using a Rayleigh fractionation model, resulting in a Ca isotope fractionation factor (Δ⁴⁴Ca) equal to −0.72 ± 0.05‰. Numerical modeling suggests that Ca uptake by these cyanobacteria is primarily unidirectional, with minimal back reaction observed over the duration of the experiment. Finally, we compared our Δ⁴⁴Ca values with those of other biotic and abiotic carbonates, revealing similarities with organisms that form biogenic calcite. These similarities raise questions about the effectiveness of using the Ca isotope fractionation factor as a univocal tracer of ACC-forming cyanobacteria in the environment. We propose that the use of Δ⁴⁴Ca in combination with other proposed tracers of ACC-forming cyanobacteria such as Ba and Sr isotope fractionation factors and/or elevated Ba/Ca and Sr/Ca ratios may provide a more reliable approach.     

Serum rapidly mobilizes calcium from an intracellular pool in quiescent fibroblastic cells

Addition of dialysed fetal bovine serum to quiescent cultures of Swiss 3T3 cells loaded with 45Ca2+ causes a very rapid increase in the rate of 45Ca2+ efflux from an intracellular pool. Exposure to serum for 2 min leads to a fall of 0.59 nmol Ca2+/mg protein in the intracellular Ca2+ content of the cells. Inhibitors of mitochondrial function prevent the stimulation of 45Ca2+ efflux by serum. The stimulation of 45Ca2+ efflux by serum is also observed in quiescent cultures of Rat-1, Swiss 3T6 and BHK cells and in secondary cultures of whole mouse embryo fibroblasts.