RESUMO
Metabolic adaptation may happen in response to the pressure exerted by the microenvironment and is a key step in survival of metastatic cells. Brain metastasis occurs as a consequence of the systemic dissemination of tumor cells, a fact that correlates with poor prognosis and high morbidity due to the difficulty in identifying biomarkers that allow a more targeted therapy. Previously, we performed transcriptomic analysis of human breast cancer patient samples and evaluated the differential expression of genes in brain metastasis (BrM) compared to lung, bone and liver metastasis. Our network approach identified upregulation of glucose-regulated protein 94 (GRP94) as well as proteins related to synthesis of fatty acids (FA) in BrM. Here we report that BrM cells show an increase in FA content and decreased saturation with regard to parental cells measured by Raman spectroscopy that differentiate BrM from other metastases. Moreover, BrM cells exerted a high ability to oxidize FA and compensate hypoglycemic stress due to an overexpression of proteins involved in FA synthesis and degradation (SREBP-1, LXRα, ACOT7). GRP94 ablation restored glucose dependence, down-regulated ACOT7 and SREBP-1 and decreased tumorigenicity in vivo. In conclusion, GRP94 is required for the metabolic stress survival of BrM cells, and it might act as a modulator of lipid metabolism to favor BrM progression.
Assuntos
Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Ácidos Graxos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Ácidos Graxos/análise , Feminino , Proteínas de Choque Térmico HSP70/análise , Humanos , Proteínas de Membrana/análise , Camundongos NusRESUMO
Raman spectroscopy (RS) has shown promise as a tool to reveal biochemical changes that occur in cancer processes at the cellular level. However, when analyzing clinical samples, RS requires improvements to be able to resolve biological components from the spectra. We compared the strengths of Multivariate Curve Resolution (MCR) versus Principal Component Analysis (PCA) to deconvolve meaningful biological components formed by distinct mixtures of biological molecules from a set of mixed spectra. We exploited the flexibility of the MCR algorithm to easily accommodate different initial estimates and constraints. We demonstrate the ability of MCR to resolve undesired background signals from the RS that can be subtracted to obtain clearer cancer cell spectra. We used two triple negative breast cancer cell lines, MDA-MB 231 and MDA-MB 435, to illustrate the insights obtained by RS that infer the metabolic changes required for metastasis progression. Our results show that increased levels of amino acids and lower levels of mitochondrial signals are attributes of bone metastatic cells, whereas lung metastasis tropism is characterized by high lipid and mitochondria levels. Therefore, we propose a method based on the MCR algorithm to achieve unique biochemical insights into the molecular progression of cancer cells using RS.
Assuntos
Algoritmos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias da Mama/metabolismo , Neoplasias Ósseas/patologia , Neoplasias da Mama/patologia , Feminino , Humanos , Análise Espectral Raman , Células Tumorais CultivadasRESUMO
Receptor Tyrosine Kinase (RTK) signaling pathways induce multiple biological responses, often by regulating the expression of downstream genes. The HMG-box protein Capicua (Cic) is a transcriptional repressor that is downregulated in response to RTK signaling, thereby enabling RTK-dependent induction of Cic targets. In both Drosophila and mammals, Cic is expressed as two isoforms, long (Cic-L) and short (Cic-S), whose functional significance and mechanism of action are not well understood. Here we show that Drosophila Cic relies on the Groucho (Gro) corepressor during its function in the early embryo, but not during other stages of development. This Gro-dependent mechanism requires a short peptide motif, unique to Cic-S and designated N2, which is distinct from other previously defined Gro-interacting motifs and functions as an autonomous, transferable repressor element. Unexpectedly, our data indicate that the N2 motif is an evolutionary innovation that originated within dipteran insects, as the Cic-S isoform evolved from an ancestral Cic-L-type form. Accordingly, the Cic-L isoform lacking the N2 motif is completely inactive in early Drosophila embryos, indicating that the N2 motif endowed Cic-S with a novel Gro-dependent activity that is obligatory at this stage. We suggest that Cic-S and Gro coregulatory functions have facilitated the evolution of the complex transcriptional network regulated by Torso RTK signaling in modern fly embryos. Notably, our results also imply that mammalian Cic proteins are unlikely to act via Gro and that their Cic-S isoform must have evolved independently of fly Cic-S. Thus, Cic proteins employ distinct repressor mechanisms that are associated with discrete structural changes in the evolutionary history of this protein family.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Padronização Corporal/genética , Proteínas de Drosophila/genética , Proteínas HMGB/genética , Receptores Proteína Tirosina Quinases/genética , Proteínas Repressoras/genética , Animais , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/metabolismo , Isoformas de Proteínas/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/genéticaAssuntos
Terapia a Laser/métodos , Lasers de Gás/uso terapêutico , Rinofima/cirurgia , Humanos , Terapia a Laser/efeitos adversos , Terapia a Laser/instrumentação , Masculino , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Rinofima/diagnóstico , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
RTK/Ras/MAPK signaling pathways play key functions in metazoan development, but how they control expression of downstream genes is not well understood. In Drosophila, it is generally assumed that most transcriptional responses to RTK signal activation depend on binding of Ets-family proteins to specific cis-acting sites in target enhancers. Here, we show that several Drosophila RTK pathways control expression of downstream genes through common octameric elements that are binding sites for the HMG-box factor Capicua, a transcriptional repressor that is downregulated by RTK signaling in different contexts. We show that Torso RTK-dependent regulation of terminal gap gene expression in the early embryo critically depends on Capicua octameric sites, and that binding of Capicua to these sites is essential for recruitment of the Groucho co-repressor to the huckebein enhancer in vivo. We then show that subsequent activation of the EGFR RTK pathway in the neuroectodermal region of the embryo controls dorsal-ventral gene expression by downregulating the Capicua protein, and that this control also depends on Capicua octameric motifs. Thus, a similar mechanism of RTK regulation operates during subdivision of the anterior-posterior and dorsal-ventral embryonic axes. We also find that identical DNA octamers mediate Capicua-dependent regulation of another EGFR target in the developing wing. Remarkably, a simple combination of activator-binding sites and Capicua motifs is sufficient to establish complex patterns of gene expression in response to both Torso and EGFR activation in different tissues. We conclude that Capicua octamers are general response elements for RTK signaling in Drosophila.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Drosophila/genética , Proteínas HMGB/genética , Sistema de Sinalização das MAP Quinases , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Repressoras/genética , Elementos de Resposta , Animais , Sítios de Ligação , Padronização Corporal , Drosophila , Receptores ErbB/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Multimerização Proteica , Asas de Animais/crescimento & desenvolvimentoRESUMO
Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species.
Assuntos
Apoptose , Blattellidae/embriologia , Ecdisterona/fisiologia , Hormônios Juvenis/fisiologia , Metamorfose Biológica , Animais , Proteínas de Ligação a DNA/fisiologia , Proteínas Inibidoras de Apoptose/fisiologia , Proteínas de Insetos/fisiologia , Ninfa/fisiologia , Receptores de Esteroides/fisiologia , Fator Esteroidogênico 1/fisiologiaRESUMO
In the absence of hormone the ecdysteroid receptor (EcR) is distributed between the cytoplasm and the nucleus. Addition of the hormone muristerone A increases nuclear localization of wild type EcR within 5-10 min. Mutation of M504 to alanine, an amino acid, which is essential for ligand binding and which is situated in helix 5 of the ligand binding domain, abolishes hormone binding but still allows nuclear localization at only slightly reduced levels in the absence of hormone, whereas nuclear localization of EcR(M504R) is nearly abolished. Cotransfection with ultraspiracle (USP), the invertebrate ortholog of RXR, leads to exclusively nuclear localization of wild type EcR and EcR(M504A) indicating that basal heterodimerization in the absence of hormone is still possible. In the presence of Usp, EcR(M504R) is only partially localized in the nucleus. EMSA experiments show that the ligand muristerone A enhances binding of wild type EcR, but only slightly of mutated EcRs, to the canonical hsp 27 ecdysone response element. This is confirmed by transactivation studies. The results indicate that the architecture of the E-domain of EcR is important for nuclear localization even in the absence of a ligand.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Ecdisterona/análogos & derivados , Receptores de Esteroides/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Ecdisterona/metabolismo , Ligantes , Mutação , Estrutura Terciária de Proteína , Receptores de Esteroides/química , Receptores de Esteroides/genética , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , TransfecçãoRESUMO
Ecdysteroids coordinate development, reproduction and other essential biological processes in insects and other arthropods through the receptor which is a heterodimer of two members of the nuclear receptors superfamily, the ecdysteroid receptor (EcR) and the Ultraspiracle (Usp). Although the transcriptionally active EcR/Usp heterocomplex is believed to be the only functional form of the receptor, there are data indicating that EcR may be involved in the mediation of the non-genomic effects outside of the nucleus. Since the nucleocytoplasmic shuttling could be a key element determining participation of the single nuclear receptor molecule both in the genomic and non-genomic functions we have analyzed nuclear import and export properties of the EcR and Usp from Drosophila melanogaster. We show for the first time that both receptors exhibit differential distribution of the nuclear localization and nuclear export signals (NLSs and NESs). In particular, the Usp which exhibits exclusively nuclear localization in all cell types analyzed, contains apparently only NLS activity within the DNA-binding domain. In contrast, the three known EcR isoforms (A, B1 and B2) are mosaics of elements which can potentially mediate their nucleocytoplasmic shuttling. We have found two active NESs in ligand binding domain and NLS activity within the DNA-binding domain of all isoforms. Simultaneously we demonstrate that B1 and A isoforms possess an additional NLS activity localized in AB regions. We speculate that this characteristic, along with the previously reported structural pliability of the EcR molecule, allows the single receptor to evoke many different genomic as well as non-genomic ecdysteroid-dependent responses.
Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila melanogaster , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Sinais de Exportação Nuclear/genética , Sinais de Exportação Nuclear/fisiologia , Sinais de Localização Nuclear/metabolismo , Receptores de Esteroides/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares , Fatores de Transcrição/genéticaRESUMO
Initially, nuclear import of the ecdysteroid receptor (EcR) in vertebrate cells (CHO-K1 and COS-7) does not afford a heterodimerization partner. Later on, EcR is retained in the nucleus only in the presence of a heterodimerization partner. Ultraspiracle (Usp) is more efficient compared to its vertebrate orthologue RXR and leads to an exclusively nuclear localization of EcR even in the absence of ligand. The DNA binding domain of the heterodimerization partner is important for retainment of EcR in the nucleus as shown by Usp4 (Usp(R130C)), which has lost its DNA binding capability. The C-terminal end of Usp (Usp(Delta205-508)) encompassing the C-terminal part of the D-domain and the E- and F-domains are essential for retainment of EcR in the nucleus. Nuclear localization is further influenced by cell-specific factors, since hormone and heterodimerization stabilizes the EcR protein in a cell-specific way.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , DNA/metabolismo , Dimerização , Proteínas de Drosophila , Domínios e Motivos de Interação entre Proteínas/fisiologia , Receptor X Retinoide alfa/metabolismo , Transcrição GênicaRESUMO
In insects, the transition from juvenile development to the adult stage is controlled by juvenile hormone (JH) synthesized from the corpora allata (CA) glands. Whereas a JH-free period during the last juvenile instar triggers metamorphosis and the end of the growth period, the reappearance of this hormone after the imaginal molt marks the onset of reproductive adulthood. Despite the importance of such transition, the regulatory mechanism that controls it remains mostly unknown. Here, using the hemimetabolous insect Blattella germanica, we show that nuclear hormone receptors Seven-up-B (BgSvp-B) and Fushi tarazu-factor 1 (BgFTZ-F1) have essential roles in the tissue- and stage-specific activation of adult CA JH-biosynthetic activity. Both factors are highly expressed in adult CA cells. Moreover, RNAi-knockdown of either BgSvp-B or BgFTZ-F1 results in adult animals with a complete block in two critical JH-dependent reproductive processes, vitellogenesis and oogenesis. We show that this reproductive blockage is the result of a dramatic impairment of JH biosynthesis, due to the CA-specific reduction in the expression of two key JH biosynthetic enzymes, 3-hydroxy-3-methylglutaryl coenzyme A synthase-1 (BgHMG-S1) and HMG-reductase (BgHMG-R). Our findings provide insights into the regulatory mechanisms underlying the specific changes in the CA gland necessary for the proper transition to adulthood.
Assuntos
Blattellidae/metabolismo , Proteínas de Insetos/metabolismo , Hormônios Juvenis/biossíntese , Receptores Citoplasmáticos e Nucleares/metabolismo , Fator Esteroidogênico 1/metabolismo , Animais , Blattellidae/crescimento & desenvolvimento , Proliferação de Células , Corpora Allata/metabolismo , Feminino , Isoformas de Proteínas/metabolismo , ReproduçãoRESUMO
Although molecular classification brings interesting insights into breast cancer taxonomy, its implementation in daily clinical care is questionable because of its expense and the information supplied in a single sample allocation is not sufficiently reliable. New approaches, based on a panel of small molecules derived from the global or targeted analysis of metabolic profiles of cells, have found a correlation between activation of de novo lipogenesis and poorer prognosis and shorter disease-free survival for many tumors. We hypothesized that the lipid content of breast cancer cells might be a useful indirect measure of a variety of functions coupled to breast cancer progression. Raman microspectroscopy was used to characterize metabolism of breast cancer cells with different degrees of malignancy. Raman spectra from MDA-MB-435, MDA-MB-468, MDA-MB-231, SKBR3, MCF7 and MCF10A cells were acquired with an InVia Raman microscope (Renishaw) with a backscattered configuration. We used Principal Component Analysis and Partial Least Squares Discriminant Analyses to assess the different profiling of the lipid composition of breast cancer cells. Characteristic bands related to lipid content were found at 3014, 2935, 2890 and 2845 cm(-1), and related to lipid and protein content at 2940 cm(-1). A classificatory model was generated which segregated metastatic cells and non-metastatic cells without basal-like phenotype with a sensitivity of 90% and a specificity of 82.1%. Moreover, expression of SREBP-1c and ABCA1 genes validated the assignation of the lipid phenotype of breast cancer cells. Indeed, changes in fatty acid unsaturation were related with the epithelial-to-mesenchymal transition phenotype. Raman microspectroscopy is a promising technique for characterizing and classifying the malignant phenotype of breast cancer cells on the basis of their lipid profiling. The algorithm for the discrimination of metastatic ability is a first step towards stratifying breast cancer cells using this rapid and reagent-free tool.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Metabolismo dos Lipídeos , Análise Espectral Raman , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Análise Discriminante , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Ácidos Graxos/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Análise dos Mínimos Quadrados , Metabolismo dos Lipídeos/genética , Microespectrofotometria , Metástase Neoplásica , Fenótipo , Análise de Componente PrincipalRESUMO
Postembryonic development of holometabolous and hemimetabolous insects occurs through successive molts triggered by 20-hydroxyecdysone (20E). The molecular action of 20E has been extensively studied in holometabolous insects, but data on hemimetabolous are scarce. We have demonstrated that during the nymphal development of the hemimetabolous insect Blattella germanica, 20E binds to the heterodimeric receptor formed by the nuclear receptors BgEcR-A and BgRXR activating a cascade of gene expression, including the nuclear receptors BgE75 and BgHR3. Herein, we report the characterization of BgFTZ-F1, another nuclear hormone receptor involved in 20E action. BgFTZ-F1 is activated at the end of each instar, and RNAi has demonstrated that BgHR3 is needed for BgFTZ-F1 activation, and that BgFTZ-F1 has critical functions of during the last nymphal instar. Nymphs with silenced BgFTZ-F1 cannot ecdyse, arrest development, and show structures of ectodermal origin duplicated. BgFTZ-F1 also controls the timing of the ecdysteroid molting pulse.
Assuntos
Blattellidae/embriologia , Proteínas de Ligação a DNA/biossíntese , Ecdisteroides/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Insetos/biossíntese , Animais , Blattellidae/genética , Proteínas de Ligação a DNA/genética , Ecdisteroides/genética , Proteínas de Insetos/genética , Muda/fisiologia , Ninfa/genética , Ninfa/metabolismoRESUMO
The small G protein Ran, which is important for nucleocytoplasmic shuttling of proteins is present, but does not interact with EcR, Usp, and EcR/Usp. As shown by oligomycin treatment, EcR, Usp, and EcR/Usp import is energy dependent. Export of EcR and EcR/Usp is mediated by exportin-1 (CRM-1) as shown by the inhibiting effect of leptomycin B (LMB). Usp remains in the nucleus for more than 24 h. Nuclear retainment of EcR and Usp is energy dependent as shown by treatment with oligomycin. No export signal could be identified for Usp. The data confirm that EcR and Usp can enter the nucleus independently and that intracellular localization is regulated individually for each receptor. It is also demonstrated that the export signal of EcR is inaccessible after heterodimerization with Usp.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster , Metabolismo Energético/fisiologia , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Drosophila , Ácidos Graxos Insaturados , Células HeLa , Humanos , Oligomicinas , Ligação Proteica , Proteína Exportina 1RESUMO
The plant hormone abscisic acid regulates gene expression in response to growth stimuli and abiotic stress. Previous studies have implicated members of the bZIP family of transcription factors as mediators of abscisic acid dependent gene expression through the ABRE cis-element. Here, we identify two new maize bZIP transcription factors, EmBP-2 and ZmBZ-1 related to EmBP-1 and OsBZ-8 families. They are differentially expressed during embryo development; EmBP-2 is constitutive, whereas ZmBZ-1 is abscisic acid-inducible and accumulates during late embryogenesis. Both factors are nuclear proteins that bind to ABREs and activate transcription of the abscisic acid-inducible gene rab28 from maize. EmBP-2 and ZmBZ-1 are phosphorylated by protein kinase CK2 and phosphorylation alters their DNA binding properties. Our data suggest that EmBP-2 and ZmBZ-1 are involved in the expression of abscisic acid inducible genes such as rab28 and their activity is modulated by ABA and by phosphorylation.
Assuntos
Ácido Abscísico/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/genética , Zea mays/genética , Proteínas rab de Ligação ao GTP/genética , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica/química , Caseína Quinase II/metabolismo , Clonagem Molecular , Dados de Sequência Molecular , Fosforilação , Folhas de Planta/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Elementos de Resposta/genética , Sementes/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ativação Transcricional/genéticaRESUMO
The heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (USP), both of which are members of the nuclear receptor superfamily, is considered to be the functional ecdysteroid receptor. Here we analyzed the subcellular distribution of EcR and USP fused to fluorescent proteins. The experiments were carried out in mammalian COS-7, CHO-K1 and HeLa cells to facilitate investigation of the subcellular trafficking of EcR and USP in the absence of endogenous expression of these two receptors. The distribution of USP tagged with a yellow fluorescent protein (YFP-USP) was almost exclusively nuclear in all cell types analyzed. The nuclear localization remained constant for at least 1 day after the first visible signs of expression. In contrast, the intracellular distribution of EcR tagged with a yellow fluorescent protein (YFP-EcR) varied and was dependent on time and cell type, although YFP-EcR alone was also able to partially translocate into the nuclear compartment. Coexpression of YFP-EcR with USP tagged with a cyan fluorescent protein (CFP-USP) resulted in exclusively nuclear localization of both proteins in all cell types analyzed. The USP-induced nuclear localization of YFP-EcR was stable for at least 20 hours. These experiments suggest that USP has a profound effect on the subcellular distribution of EcR.