We transfected HeLa GFP-AR cells using a plasmid encoding hcRED (a 25 kDa-red fluorescent proteins) to permit visualization from the cytoplasmic area (Supplementary Fig. with reduced maximal replies. HeLa cells had been transiently transfected with either untagged GFP-AR or AR plasmids as well as the pARR-2PB-dsRED reporter plasmid. Twenty-four hours after transfection, cells had been subjected to multiple concentrations of DHT which range from 0.002 nM to 200 nM for 18 hr. Cells had been then set and probed with an anti-AR antibody to visualize both GFP-tagged and untagged AR in pictures captured with the computerized IC100 microscope. Pictures had been examined using Pipeline Pilot software program as well as the nuclear translocation (A), nuclear hyperspeckling (B), and transcriptional activity (C) replies quantified.(0.83 MB TIF) pone.0003605.s002.tif (814K) GUID:?D3E323CB-0Compact disc4-4CF8-B1F9-AB516B3E0EDF Amount S3: GFP-AR retains a nuclear distribution with reduced hyperspeckling but maintains the capability to shuttle in to the cytoplasm following agonist removal. A. HeLa GFP-AR cells had been treated with 1 nM for 30 min, 1 hr, or 2 hrs. After ligand treatment, R1881 was taken out by serial washes with ligand free of charge media filled with cyclohexamide to avoid new proteins synthesis. Cells were fixed then, imaged, and analyzed for the localization from the receptor at 3, 6, 9, 12, and 15 hrs using described image analysis tools previously. Replies were normalized to untreated response and handles seen with 1 nM R1881 treatment for 2 hrs. An additional test using untagged AR was also performed to make sure response had not been because of the inclusion from the GFP label over the receptor. The power of GFP-AR to shuttle between your nuclear and cytoplasmic compartments after and during ligand treatment was analyzed using the Turn photobleaching technique in which a area in the cytoplasm is normally frequently bleached. B. A graph evaluating the speed of which nuclear GFP-AR fluorescence is normally dropped in the lack of ligand (neglected, t1/2?=?11418.1 sec, n?=?11), in the current presence of 10 nM R1881 (Treated, t1/2?=?61251.9 sec, n?=?11), and after ligand withdrawal (Withdrawal, t1/2?=?55943.2 sec, n?=?10). To make sure results weren’t because of general photobleaching during imaging, cells had been examined where in fact the targeted photobleaching area was beyond the cellular region (Photobleach Control). Both R1881 treatment and drawback significantly gradual but will not stop the speed which the receptor shuttles between your nucleus as well as the cytoplasm. C. Preferred images from Turn test.(2.19 MB TIF) pone.0003605.s003.tif (2.0M) GUID:?5B982B7E-8713-4A1C-AF72-042B658DFB07 Figure S4: Differential responses from the T877A AR mutation. The differential ramifications of the T877A mutation on AR nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity in HeLa GFP-AR with chosen substances. Cells stably expressing LY-2584702 tosylate salt either WT (unhatched) or T877A (hatched) types of AR had been transfected with pARR-2PB-dsRED2skl reporter vector and preserved in 5% SD-FBS mass media for 12 hr. Cells had been treated with indicated substance either by itself (grey pubs) or with 10 nM R1881 (white pubs) for 18 hr in 5%SD-FBS. Outcomes normalized to detrimental (no treatment) and positive (R1881) handles. When possible, EC50 beliefs were calculated using SigmaPlot 4-parameter curve fitting presentedstd and tool. mistake. Data represents typical of 4 tests.(1.51 MB TIF) pone.0003605.s004.tif (1.4M) GUID:?63743940-E3D0-464E-910F-BEE7A7EC5EDF Desk S1: (0.05 MB PDF) pone.0003605.s005.pdf (46K) GUID:?DDC8A4B8-BD5B-4E53-A4E7-AC5B78AE324A Desk S2: (0.03 MB PDF) pone.0003605.s006.pdf (31K) GUID:?40ECC376-BB21-4A38-A709-2F1CBDDF71AE Desk S3: (0.04 MB PDF) pone.0003605.s007.pdf (36K) GUID:?4865083F-B744-45EB-97E8-4D41E3DFAF23 Desk S4: (0.03 MB PDF) pone.0003605.s008.pdf (26K) GUID:?2DFCD190-CDD3-481F-9DF5-78BAdvertisement06B4BFD Desk S5: (0.56 MB TIF) pone.0003605.s009.tif (547K) GUID:?C5CDECD2-52AF-4B3C-9C68-9112901024CF Desk S6: (0.06 MB PDF) pone.0003605.s010.pdf (60K) GUID:?0F47AFE9-6C95-4AF6-B34D-00EE1B88520A Desk S7: (0.04 MB PDF) pone.0003605.s011.pdf (42K) GUID:?EAF11A2E-B302-4B87-BA60-F4B1649E3D0D Abstract History Focusing on how androgen receptor (AR) function is normally modulated by contact with steroids, growth elements or little molecules may have essential mechanistic implications for AR-related disease therapies (e.g., prostate cancers, androgen insensitivity symptoms, AIS), and in the evaluation of environmental endocrine disruptors. Technique/Principal Results We report the introduction of a higher throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on the cell-by-cell basis. Furthermore, simultaneous evaluation of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variance were 5C24%, with Z’ values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, exhibited that nuclear translocation and nuclear speckling were linked with transcriptional output, and specific ligands were noted to differentially impact measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations exhibited a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions. Conclusions/Significance HT imaging-based multiplex.This tessellation in combination with a user defined radius rule defines cytoplasmic compartment of each cell. exposed to multiple concentrations of DHT ranging from 0.002 nM to 200 nM for 18 hr. Cells were then fixed and probed with an anti-AR antibody to visualize both GFP-tagged and untagged AR in images captured by the automated IC100 microscope. Images were analyzed using Pipeline Pilot software and the nuclear translocation (A), nuclear hyperspeckling (B), and transcriptional activity (C) responses quantified.(0.83 MB TIF) pone.0003605.s002.tif (814K) GUID:?D3E323CB-0CD4-4CF8-B1F9-AB516B3E0EDF Physique S3: GFP-AR retains a nuclear distribution with decreased hyperspeckling but maintains the ability to shuttle into the cytoplasm after agonist removal. A. HeLa GFP-AR cells were treated with 1 nM for 30 min, 1 hr, or 2 hrs. After ligand treatment, R1881 was removed by serial washes with ligand free media made up of cyclohexamide to prevent new protein synthesis. Cells were then fixed, imaged, and examined for the localization of the receptor at 3, 6, 9, 12, and 15 hrs using previously explained image analysis tools. Responses were normalized to untreated controls and response seen with 1 nM R1881 treatment for 2 hrs. An additional experiment using untagged AR was also performed to ensure response was not due to the inclusion of the GFP tag around the receptor. The ability of GFP-AR to shuttle between the nuclear and cytoplasmic compartments during and after ligand treatment was analyzed using the FLIP photobleaching technique where a region in the cytoplasm is usually repeatedly bleached. B. A graph comparing the rate at which nuclear GFP-AR fluorescence is usually lost in the absence of ligand (untreated, t1/2?=?11418.1 sec, n?=?11), in the presence of 10 nM R1881 (Treated, t1/2?=?61251.9 sec, n?=?11), and after ligand withdrawal (Withdrawal, t1/2?=?55943.2 sec, n?=?10). To ensure results were not LY-2584702 tosylate salt due to general photobleaching during imaging, cells were examined where the targeted photobleaching region was outside of the cellular area (Photobleach Control). Both R1881 treatment and withdrawal significantly slow but does not stop the rate that this receptor shuttles between the nucleus and the cytoplasm. C. Determined images from FLIP experiment.(2.19 MB TIF) pone.0003605.s003.tif (2.0M) GUID:?5B982B7E-8713-4A1C-AF72-042B658DFB07 Figure S4: Differential responses of the T877A AR mutation. The differential effects of the T877A mutation on AR nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity in HeLa GFP-AR with selected compounds. Cells stably expressing either WT (unhatched) or T877A (hatched) forms of AR were transfected with pARR-2PB-dsRED2skl reporter vector and managed in 5% SD-FBS media for 12 hr. Cells were treated with indicated compound either alone (grey bars) or with 10 nM R1881 (white bars) for 18 hr in 5%SD-FBS. Results normalized to unfavorable (no treatment) and positive (R1881) controls. When possible, EC50 values were calculated using SigmaPlot 4-parameter curve fitted tool and presentedstd. error. Data represents average of 4 experiments.(1.51 MB TIF) pone.0003605.s004.tif (1.4M) GUID:?63743940-E3D0-464E-910F-BEE7A7EC5EDF Table S1: (0.05 MB PDF) pone.0003605.s005.pdf (46K) GUID:?DDC8A4B8-BD5B-4E53-A4E7-AC5B78AE324A Table S2: (0.03 MB PDF) pone.0003605.s006.pdf (31K) GUID:?40ECC376-BB21-4A38-A709-2F1CBDDF71AE Table S3: (0.04 MB PDF) pone.0003605.s007.pdf (36K) GUID:?4865083F-B744-45EB-97E8-4D41E3DFAF23 Table S4: (0.03 MB PDF) pone.0003605.s008.pdf (26K) GUID:?2DFCD190-CDD3-481F-9DF5-78BAD06B4BFD Table S5: (0.56 MB TIF) pone.0003605.s009.tif (547K) GUID:?C5CDECD2-52AF-4B3C-9C68-9112901024CF Table S6: (0.06 MB PDF) pone.0003605.s010.pdf (60K) GUID:?0F47AFE9-6C95-4AF6-B34D-00EE1B88520A Table S7: (0.04 MB PDF) pone.0003605.s011.pdf (42K) GUID:?EAF11A2E-B302-4B87-BA60-F4B1649E3D0D Abstract Background Understanding how androgen receptor (AR) function is usually modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate malignancy, androgen insensitivity syndrome, AIS), and in the analysis of environmental endocrine disruptors. Methodology/Principal Findings We report the development of a.No funding sources had any role in the collection, analysis, and interpretation of the data, and in LY-2584702 tosylate salt the preparation, review, or approval of the manuscript.. with either untagged AR or GFP-AR plasmids in addition to the pARR-2PB-dsRED reporter plasmid. Twenty-four hours after transfection, cells were exposed to multiple concentrations of DHT ranging from 0.002 nM to 200 nM for 18 hr. Cells were then fixed and probed with an anti-AR antibody to visualize both GFP-tagged and untagged AR in images captured by the automated IC100 microscope. Images were analyzed using Pipeline Pilot software and the nuclear translocation (A), nuclear hyperspeckling (B), and transcriptional activity (C) responses quantified.(0.83 MB TIF) pone.0003605.s002.tif (814K) GUID:?D3E323CB-0CD4-4CF8-B1F9-AB516B3E0EDF Physique S3: GFP-AR retains a nuclear distribution with decreased hyperspeckling but maintains the ability to shuttle into the cytoplasm after agonist removal. A. HeLa GFP-AR cells were treated with 1 nM for 30 min, 1 hr, or 2 hrs. After ligand treatment, R1881 was removed by serial washes with ligand free media containing cyclohexamide to prevent new protein synthesis. Cells were then fixed, imaged, and examined for the localization of the receptor at 3, 6, 9, 12, and 15 hrs using previously described image analysis tools. Responses were normalized to untreated controls and response seen with 1 nM R1881 treatment for 2 hrs. An additional experiment using untagged AR was also performed to ensure response was not due to the inclusion of the GFP tag on the receptor. The ability of GFP-AR to shuttle between the nuclear and cytoplasmic compartments during and after ligand treatment was analyzed using the FLIP photobleaching technique where a region in the cytoplasm is repeatedly bleached. B. A graph comparing the rate at which nuclear GFP-AR fluorescence is lost in the absence of ligand (untreated, t1/2?=?11418.1 sec, n?=?11), in the presence of 10 nM R1881 (Treated, t1/2?=?61251.9 sec, n?=?11), and after ligand withdrawal (Withdrawal, t1/2?=?55943.2 sec, n?=?10). To ensure results were not due to general photobleaching during imaging, cells were examined where the targeted photobleaching region was outside of the cellular area (Photobleach Control). Both R1881 treatment and withdrawal significantly slow but does not stop the rate that the receptor shuttles between the nucleus and the cytoplasm. C. Selected images from FLIP experiment.(2.19 MB TIF) pone.0003605.s003.tif (2.0M) GUID:?5B982B7E-8713-4A1C-AF72-042B658DFB07 Figure S4: Differential responses of the T877A AR mutation. The differential effects of the T877A mutation on AR nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity in HeLa GFP-AR with selected compounds. Cells stably expressing either WT (unhatched) or T877A (hatched) forms of AR were transfected with pARR-2PB-dsRED2skl reporter vector and maintained in 5% SD-FBS media for 12 hr. Cells were treated with indicated compound either alone (grey bars) or with 10 nM R1881 (white bars) for 18 hr in 5%SD-FBS. Results normalized to negative (no treatment) and positive (R1881) controls. When possible, EC50 values were calculated using SigmaPlot 4-parameter curve fitting tool and presentedstd. error. Data represents average of 4 experiments.(1.51 MB TIF) pone.0003605.s004.tif (1.4M) GUID:?63743940-E3D0-464E-910F-BEE7A7EC5EDF Table S1: (0.05 MB PDF) pone.0003605.s005.pdf (46K) GUID:?DDC8A4B8-BD5B-4E53-A4E7-AC5B78AE324A Table S2: (0.03 MB PDF) pone.0003605.s006.pdf (31K) GUID:?40ECC376-BB21-4A38-A709-2F1CBDDF71AE Table S3: (0.04 MB PDF) pone.0003605.s007.pdf (36K) GUID:?4865083F-B744-45EB-97E8-4D41E3DFAF23 Table S4: (0.03 MB PDF) pone.0003605.s008.pdf (26K) GUID:?2DFCD190-CDD3-481F-9DF5-78BAD06B4BFD Table S5: (0.56 MB TIF) pone.0003605.s009.tif (547K) GUID:?C5CDECD2-52AF-4B3C-9C68-9112901024CF Table S6: (0.06 MB PDF) pone.0003605.s010.pdf (60K) GUID:?0F47AFE9-6C95-4AF6-B34D-00EE1B88520A Table S7: (0.04 MB PDF) pone.0003605.s011.pdf (42K) GUID:?EAF11A2E-B302-4B87-BA60-F4B1649E3D0D Abstract Background Understanding how androgen receptor (AR) function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS), and in the analysis of environmental endocrine disruptors. Methodology/Principal Findings We report the development of a high throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the.Results normalized to negative (no treatment) and positive (R1881) controls. plate of the set.(2.38 MB TIF) pone.0003605.s001.tif (2.2M) GUID:?8F9D2408-566C-4322-911C-78031CB32B72 Figure S2: GFP-AR responds in a similar manner to untagged AR but with diminished maximal responses. HeLa cells were transiently transfected with either untagged AR or GFP-AR plasmids in addition to the pARR-2PB-dsRED reporter plasmid. Twenty-four hours after transfection, cells were exposed to multiple concentrations of DHT ranging from 0.002 nM to 200 nM for 18 hr. Cells were then fixed and probed with an anti-AR antibody to visualize both GFP-tagged and untagged AR in images captured by the automated IC100 microscope. Images were analyzed using Pipeline Pilot software and the nuclear translocation (A), nuclear hyperspeckling (B), and transcriptional activity (C) responses quantified.(0.83 MB TIF) pone.0003605.s002.tif (814K) GUID:?D3E323CB-0CD4-4CF8-B1F9-AB516B3E0EDF Figure S3: GFP-AR retains a nuclear distribution with decreased hyperspeckling but maintains the ability to shuttle into the cytoplasm after agonist removal. A. HeLa GFP-AR cells were treated with 1 nM for 30 min, 1 hr, or 2 hrs. After ligand treatment, R1881 was removed by serial washes with ligand free media containing cyclohexamide to prevent new protein synthesis. Cells were then fixed, imaged, and examined for the localization of the receptor at 3, 6, 9, 12, and 15 hrs using previously described image analysis tools. Responses were normalized to neglected settings and response noticed with 1 nM R1881 treatment for 2 hrs. Yet another test using untagged AR was also performed to make sure response had not been because of the inclusion from the GFP label for the receptor. The power of GFP-AR to shuttle between your nuclear and cytoplasmic compartments after and during ligand treatment was analyzed using the Turn photobleaching technique in which a area in the cytoplasm can be frequently bleached. B. A graph evaluating the pace of which nuclear GFP-AR fluorescence can be dropped in the lack of ligand (neglected, t1/2?=?11418.1 sec, n?=?11), in the current presence of 10 nM R1881 (Treated, LY-2584702 tosylate salt t1/2?=?61251.9 sec, n?=?11), and after ligand withdrawal (Withdrawal, t1/2?=?55943.2 sec, n?=?10). To make sure results weren’t because of general photobleaching during imaging, HDAC10 cells had been examined where in fact the targeted photobleaching area was beyond the cellular region (Photobleach Control). Both R1881 treatment and drawback significantly sluggish but will not stop the pace how the receptor shuttles between your nucleus as well as the cytoplasm. C. Decided on images from Turn test.(2.19 MB TIF) pone.0003605.s003.tif (2.0M) GUID:?5B982B7E-8713-4A1C-AF72-042B658DFB07 Figure S4: Differential responses from the T877A AR mutation. The differential ramifications of the T877A mutation on AR nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity in HeLa GFP-AR with chosen substances. Cells stably expressing either WT (unhatched) or T877A (hatched) types of AR had been transfected with pARR-2PB-dsRED2skl reporter vector and taken care of in 5% SD-FBS press for 12 hr. Cells had been treated with indicated substance either only (grey pubs) or with 10 nM R1881 (white pubs) for 18 hr in 5%SD-FBS. Outcomes normalized to adverse (no treatment) and positive (R1881) settings. When feasible, EC50 values had been determined using SigmaPlot 4-parameter curve installing device and presentedstd. mistake. Data represents typical of 4 tests.(1.51 MB TIF) pone.0003605.s004.tif (1.4M) GUID:?63743940-E3D0-464E-910F-BEE7A7EC5EDF Desk S1: (0.05 MB PDF) pone.0003605.s005.pdf (46K) GUID:?DDC8A4B8-BD5B-4E53-A4E7-AC5B78AE324A Desk S2: (0.03 MB PDF) pone.0003605.s006.pdf (31K) GUID:?40ECC376-BB21-4A38-A709-2F1CBDDF71AE Desk S3: (0.04 MB PDF) pone.0003605.s007.pdf (36K) GUID:?4865083F-B744-45EB-97E8-4D41E3DFAF23 Desk S4: (0.03 MB PDF) pone.0003605.s008.pdf (26K) GUID:?2DFCD190-CDD3-481F-9DF5-78BAdvertisement06B4BFD Desk S5: (0.56 MB TIF) pone.0003605.s009.tif (547K) GUID:?C5CDECD2-52AF-4B3C-9C68-9112901024CF Desk S6: (0.06 MB PDF) pone.0003605.s010.pdf (60K) GUID:?0F47AFE9-6C95-4AF6-B34D-00EE1B88520A Desk S7: (0.04 MB PDF) pone.0003605.s011.pdf (42K) GUID:?EAF11A2E-B302-4B87-BA60-F4B1649E3D0D Abstract History Focusing on how androgen receptor (AR) function is definitely modulated by contact with steroids, growth elements or little molecules may have essential mechanistic implications for AR-related disease therapies (e.g., prostate tumor, androgen insensitivity symptoms, AIS), and in the evaluation of environmental endocrine disruptors. Strategy/Principal Results We report the introduction of a higher throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on the cell-by-cell basis. Furthermore, simultaneous evaluation of DNA.The power of GFP-AR to shuttle between your nuclear and cytoplasmic compartments after and during ligand treatment was analyzed using the FLIP photobleaching technique in which a region in the cytoplasm is repeatedly bleached. 18 hr. Cells had been then set and probed with an anti-AR antibody to visualize both GFP-tagged and untagged AR in pictures captured from the computerized IC100 microscope. Pictures had been examined using Pipeline Pilot software program as well as the nuclear translocation (A), nuclear hyperspeckling (B), and transcriptional activity (C) reactions quantified.(0.83 MB TIF) pone.0003605.s002.tif (814K) GUID:?D3E323CB-0Compact disc4-4CF8-B1F9-AB516B3E0EDF Shape S3: GFP-AR retains a nuclear distribution with reduced hyperspeckling but maintains the capability to shuttle in to the cytoplasm following agonist removal. A. HeLa GFP-AR cells had been treated with 1 nM for 30 min, 1 hr, or 2 hrs. After ligand treatment, R1881 was eliminated by serial washes with ligand free of charge media including cyclohexamide to avoid new proteins synthesis. Cells had been then set, imaged, and analyzed for the localization from the receptor at 3, 6, 9, 12, and 15 hrs using previously referred to image evaluation tools. Responses had been normalized to neglected settings and response noticed with 1 nM R1881 treatment for 2 hrs. Yet another test using untagged AR was also performed to make sure response had not been because of the inclusion from the GFP label for the receptor. The power of GFP-AR to shuttle between your nuclear and cytoplasmic compartments after and during ligand treatment was analyzed using the Turn photobleaching technique in which a area in the cytoplasm can be frequently bleached. B. A graph evaluating the pace of which nuclear GFP-AR fluorescence can be dropped in the lack of ligand (neglected, t1/2?=?11418.1 sec, n?=?11), in the current presence of 10 nM R1881 (Treated, t1/2?=?61251.9 sec, n?=?11), and after ligand withdrawal (Withdrawal, t1/2?=?55943.2 sec, n?=?10). To make sure results weren’t because of general photobleaching during imaging, cells had been examined where in fact the targeted photobleaching area was beyond the cellular region (Photobleach Control). Both R1881 treatment and drawback significantly gradual but will not stop the speed which the receptor shuttles between your nucleus as well as the cytoplasm. C. Preferred images from Turn test.(2.19 MB TIF) pone.0003605.s003.tif (2.0M) GUID:?5B982B7E-8713-4A1C-AF72-042B658DFB07 Figure S4: Differential responses from the T877A AR mutation. The differential ramifications of the T877A mutation on AR nuclear translocation, nuclear hyperspeckling, and transcriptional reporter gene activity in HeLa GFP-AR with chosen substances. Cells stably expressing either WT (unhatched) or T877A (hatched) types of AR had been transfected with pARR-2PB-dsRED2skl reporter vector and preserved in 5% SD-FBS mass media for 12 hr. Cells had been treated with indicated substance either by itself (grey pubs) or with 10 nM R1881 (white pubs) for 18 hr in 5%SD-FBS. Outcomes normalized to detrimental (no treatment) and positive (R1881) handles. When feasible, EC50 values had been computed using SigmaPlot 4-parameter curve appropriate device and presentedstd. mistake. Data represents typical of 4 tests.(1.51 MB TIF) pone.0003605.s004.tif (1.4M) GUID:?63743940-E3D0-464E-910F-BEE7A7EC5EDF Desk S1: (0.05 MB PDF) pone.0003605.s005.pdf (46K) GUID:?DDC8A4B8-BD5B-4E53-A4E7-AC5B78AE324A Desk S2: (0.03 MB PDF) pone.0003605.s006.pdf (31K) GUID:?40ECC376-BB21-4A38-A709-2F1CBDDF71AE Desk S3: (0.04 MB PDF) pone.0003605.s007.pdf (36K) GUID:?4865083F-B744-45EB-97E8-4D41E3DFAF23 Desk S4: (0.03 MB PDF) pone.0003605.s008.pdf (26K) GUID:?2DFCD190-CDD3-481F-9DF5-78BAdvertisement06B4BFD Desk S5: (0.56 MB TIF) pone.0003605.s009.tif (547K) GUID:?C5CDECD2-52AF-4B3C-9C68-9112901024CF Desk S6: (0.06 MB PDF) pone.0003605.s010.pdf (60K) GUID:?0F47AFE9-6C95-4AF6-B34D-00EE1B88520A Desk S7: (0.04 MB PDF) pone.0003605.s011.pdf (42K) GUID:?EAF11A2E-B302-4B87-BA60-F4B1649E3D0D Abstract History Focusing on how androgen receptor (AR) function is normally modulated by contact with steroids, growth elements or little molecules may have essential mechanistic implications for AR-related disease therapies (e.g., prostate cancers, androgen insensitivity symptoms, AIS), and in the evaluation of environmental endocrine disruptors. Technique/Principal Results We report the introduction of a higher throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on the cell-by-cell basis. Furthermore, simultaneous evaluation of DNA articles allowed perseverance of cell routine position and allowed the evaluation of cell routine dependent adjustments in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients.