Sured by flow cytometry. (TIF) Figure S4 Confocal microscopy of Oct4-GFP mES cellsFigure S5 The effect of GSK-J4 Sox2-MB on the mRNA level of stemness genes on treated and untreated mES cells. Cells were analyzed for (A) Sox2 and (B) Nanog mRNA expression after 1 h and 24 h of treatment with the Sox2-MB. As controls, untreated mES cells were analyzed in parallel. (n = 4 per sample, ns = not significant) Error bars represent the mean 6 SEM. (TIF) Table S1 Primers used for Real-time PCR.(TIF)AcknowledgmentsThe authors thank the Flow Cytometry Core Facility (Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland) and the Bioimaging and ??Optics Platform (Ecole Polytechnique Federale de Lausanne, Lausanne, ??Switzerland) for their assistance.treated with Sox2-MB. (A) Living Oct4-GFP mES cells treated with the Sox2-MB with orthogonal slices in the xz-plane and yzplane are shown. (B) As a control, living Oct4-GFP mES cells treated with the nonspecific-MB with orthogonal slices in the xzplane and yz-plane are shown. Scale bar = 20 mm. (TIF)Author ContributionsConceived and designed the experiments: HML MPL JAH. Performed the experiments: HML STL MR. Analyzed the data: HML MR MPL PF JAH. Contributed reagents/materials/analysis tools: HML STL MR DV PF. Wrote the paper: HML MR PF MPL JAH.
Prostate cancer (CaP) initially presents as an androgen dependent (AD) disease, but frequently progresses to an androgen depletion independent (ADI) or castration-resistant state. As the latter escapes therapies which target the androgen receptor signaling axis, considerable efforts have been made to more thoroughly understand both the transition to and biology of ADI disease. The most representative in vitro model of CaP transition from AD to ADI growth is the CWR22Rv1 cell line. Like the AD CaP cell line LNCaP, CWR22Rv1 retains a functional androgen receptor (AR) and, as such, is responsive to the presence or absence of DHT. However, in contrast to LNCaP and more in line 1676428 with advanced CaP cell lines, CWR22Rv1 is not dependent upon the presence of DHT for growth. Because of the unique niche it occupies within the collection of CaP cell lines, CWR22Rv1 has been studied extensively 24272870 within the context of acquisition of ADI growth. As expected, considerable research has focused on the CWR22Rv1 androgen receptor (AR) which has been shown to carry the common H874Y mutation [1] as well as a duplication of exon 3 [2,3]. We previously reported that CWR22Rv1 and the relapsed CWR22 variant xenograft from which it was derived express an AR with a duplication of exon 3, which is accompanied by a high level of truncated AR. These properties are not present in the original androgen-dependent CWR22 xenograft, and we suggested that the truncated receptormay be responsible for the transition to its androgen-independent state. Using antibodies targeting different regions of AR, we mapped the truncated receptor species to be the N-terminal half of the molecule, consisting of NTD and DBD [2]. Since that GSK2606414 chemical information initial characterization, the genome of CWR22Rv1 has been found to carry an intragenically duplicated AR locus [4], which may account at least in part for the generation of full-length AR (FLAR) with a duplicated exon 3 and the wide range of splice variants, although the exact mechanisms remain to be elucidated. Studies by Libertini et al [5] implicated calpain in the proteolytic cleavage of full length receptor, contributing to some of the truncated receptors. By contrast, the wor.Sured by flow cytometry. (TIF) Figure S4 Confocal microscopy of Oct4-GFP mES cellsFigure S5 The effect of Sox2-MB on the mRNA level of stemness genes on treated and untreated mES cells. Cells were analyzed for (A) Sox2 and (B) Nanog mRNA expression after 1 h and 24 h of treatment with the Sox2-MB. As controls, untreated mES cells were analyzed in parallel. (n = 4 per sample, ns = not significant) Error bars represent the mean 6 SEM. (TIF) Table S1 Primers used for Real-time PCR.(TIF)AcknowledgmentsThe authors thank the Flow Cytometry Core Facility (Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland) and the Bioimaging and ??Optics Platform (Ecole Polytechnique Federale de Lausanne, Lausanne, ??Switzerland) for their assistance.treated with Sox2-MB. (A) Living Oct4-GFP mES cells treated with the Sox2-MB with orthogonal slices in the xz-plane and yzplane are shown. (B) As a control, living Oct4-GFP mES cells treated with the nonspecific-MB with orthogonal slices in the xzplane and yz-plane are shown. Scale bar = 20 mm. (TIF)Author ContributionsConceived and designed the experiments: HML MPL JAH. Performed the experiments: HML STL MR. Analyzed the data: HML MR MPL PF JAH. Contributed reagents/materials/analysis tools: HML STL MR DV PF. Wrote the paper: HML MR PF MPL JAH.
Prostate cancer (CaP) initially presents as an androgen dependent (AD) disease, but frequently progresses to an androgen depletion independent (ADI) or castration-resistant state. As the latter escapes therapies which target the androgen receptor signaling axis, considerable efforts have been made to more thoroughly understand both the transition to and biology of ADI disease. The most representative in vitro model of CaP transition from AD to ADI growth is the CWR22Rv1 cell line. Like the AD CaP cell line LNCaP, CWR22Rv1 retains a functional androgen receptor (AR) and, as such, is responsive to the presence or absence of DHT. However, in contrast to LNCaP and more in line 1676428 with advanced CaP cell lines, CWR22Rv1 is not dependent upon the presence of DHT for growth. Because of the unique niche it occupies within the collection of CaP cell lines, CWR22Rv1 has been studied extensively 24272870 within the context of acquisition of ADI growth. As expected, considerable research has focused on the CWR22Rv1 androgen receptor (AR) which has been shown to carry the common H874Y mutation [1] as well as a duplication of exon 3 [2,3]. We previously reported that CWR22Rv1 and the relapsed CWR22 variant xenograft from which it was derived express an AR with a duplication of exon 3, which is accompanied by a high level of truncated AR. These properties are not present in the original androgen-dependent CWR22 xenograft, and we suggested that the truncated receptormay be responsible for the transition to its androgen-independent state. Using antibodies targeting different regions of AR, we mapped the truncated receptor species to be the N-terminal half of the molecule, consisting of NTD and DBD [2]. Since that initial characterization, the genome of CWR22Rv1 has been found to carry an intragenically duplicated AR locus [4], which may account at least in part for the generation of full-length AR (FLAR) with a duplicated exon 3 and the wide range of splice variants, although the exact mechanisms remain to be elucidated. Studies by Libertini et al [5] implicated calpain in the proteolytic cleavage of full length receptor, contributing to some of the truncated receptors. By contrast, the wor.
