ad rfp Search Results


adenoviral particles  (Vector Biolabs)
96
Vector Biolabs adenoviral particles
A-D. Rictor FL/FL PMECs and organoids were infected with the indicated adenoviruses. A-B. Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent <t>P-PKC-alpha</t> bands normalized to total PKC-alpha levels. C . Organoids photographed after 10 days in Matrigel culture. Average number of branches/organoid ±S.D. indicated below panels. N = 3 independent organoid isolates/condition analyzed in triplicate, Student’s T-test. D. Average colony size of organoids measured in pixel area ± S.D. N = 3 independent organoid isolates/condition, analyzed in triplicate, Student’s T-test . E. Western analysis of MCF10A parental and Rictor ZFN lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. F-I. MCF10A parental and Rictor ZFN cells were infected with Ad.RFP or Ad.PKC-alpha. F. Western analysis of MCF10A parental and Rictor ZFN lysates, 48 hours post-infection. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. G. GST-Pak-PBD effector pull-downs followed by western analysis for Rac performed 48 hours post-infection. Quantitation was performed using Image J software. Numbers in upper panel represent Rac-GTP levels in MCF10A-Rictor ZFN compared to MCF10A parental control. Lower numbers represent Rac-GTP levels normalized to total Rac for each sample. H. Cells were assessed for invasion through Matrigel-coated transwell filters. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in triplicate. I. Cells were assessed for Annexin V-FITC. Number of Annexin V-FITC+ per total number of cells was quantitated in Image J, Student’s T-test. N = 2 independent experiments, analyzed in triplicate. J. MCF10A parental cells were assessed for invasion through Matrigel-coated transwell filters in the presence of PKC-alpha inhibitor GO6976. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in quadruplicate.
Adenoviral Particles, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ad+rfp/Ad-RFP/pmc04488502-218-7-12
Average 96 stars, based on 1 article reviews
adenoviral particles - by Bioz Stars, 2026-09
96/100 stars
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cre recombinase ad cre  (Vector Biolabs)
96
Vector Biolabs cre recombinase ad cre
A-D. Rictor FL/FL PMECs and organoids were infected with the indicated adenoviruses. A-B. Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent <t>P-PKC-alpha</t> bands normalized to total PKC-alpha levels. C . Organoids photographed after 10 days in Matrigel culture. Average number of branches/organoid ±S.D. indicated below panels. N = 3 independent organoid isolates/condition analyzed in triplicate, Student’s T-test. D. Average colony size of organoids measured in pixel area ± S.D. N = 3 independent organoid isolates/condition, analyzed in triplicate, Student’s T-test . E. Western analysis of MCF10A parental and Rictor ZFN lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. F-I. MCF10A parental and Rictor ZFN cells were infected with Ad.RFP or Ad.PKC-alpha. F. Western analysis of MCF10A parental and Rictor ZFN lysates, 48 hours post-infection. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. G. GST-Pak-PBD effector pull-downs followed by western analysis for Rac performed 48 hours post-infection. Quantitation was performed using Image J software. Numbers in upper panel represent Rac-GTP levels in MCF10A-Rictor ZFN compared to MCF10A parental control. Lower numbers represent Rac-GTP levels normalized to total Rac for each sample. H. Cells were assessed for invasion through Matrigel-coated transwell filters. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in triplicate. I. Cells were assessed for Annexin V-FITC. Number of Annexin V-FITC+ per total number of cells was quantitated in Image J, Student’s T-test. N = 2 independent experiments, analyzed in triplicate. J. MCF10A parental cells were assessed for invasion through Matrigel-coated transwell filters in the presence of PKC-alpha inhibitor GO6976. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in quadruplicate.
Cre Recombinase Ad Cre, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ad+rfp/Ad-RFP-Cre/pm35671924-57-7-10
Average 96 stars, based on 1 article reviews
cre recombinase ad cre - by Bioz Stars, 2026-09
96/100 stars
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shrna rfp  (Vector Biolabs)
96
Vector Biolabs shrna rfp
A-D. Rictor FL/FL PMECs and organoids were infected with the indicated adenoviruses. A-B. Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent <t>P-PKC-alpha</t> bands normalized to total PKC-alpha levels. C . Organoids photographed after 10 days in Matrigel culture. Average number of branches/organoid ±S.D. indicated below panels. N = 3 independent organoid isolates/condition analyzed in triplicate, Student’s T-test. D. Average colony size of organoids measured in pixel area ± S.D. N = 3 independent organoid isolates/condition, analyzed in triplicate, Student’s T-test . E. Western analysis of MCF10A parental and Rictor ZFN lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. F-I. MCF10A parental and Rictor ZFN cells were infected with Ad.RFP or Ad.PKC-alpha. F. Western analysis of MCF10A parental and Rictor ZFN lysates, 48 hours post-infection. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. G. GST-Pak-PBD effector pull-downs followed by western analysis for Rac performed 48 hours post-infection. Quantitation was performed using Image J software. Numbers in upper panel represent Rac-GTP levels in MCF10A-Rictor ZFN compared to MCF10A parental control. Lower numbers represent Rac-GTP levels normalized to total Rac for each sample. H. Cells were assessed for invasion through Matrigel-coated transwell filters. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in triplicate. I. Cells were assessed for Annexin V-FITC. Number of Annexin V-FITC+ per total number of cells was quantitated in Image J, Student’s T-test. N = 2 independent experiments, analyzed in triplicate. J. MCF10A parental cells were assessed for invasion through Matrigel-coated transwell filters in the presence of PKC-alpha inhibitor GO6976. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in quadruplicate.
Shrna Rfp, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ad+rfp/Ad-U6-shRNA-RFP/pm27658230-188-11-12
Average 96 stars, based on 1 article reviews
shrna rfp - by Bioz Stars, 2026-09
96/100 stars
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ad(rgd)-rfp-icre  (Vector Biolabs)
93
Vector Biolabs ad(rgd)-rfp-icre
A-D. Rictor FL/FL PMECs and organoids were infected with the indicated adenoviruses. A-B. Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent <t>P-PKC-alpha</t> bands normalized to total PKC-alpha levels. C . Organoids photographed after 10 days in Matrigel culture. Average number of branches/organoid ±S.D. indicated below panels. N = 3 independent organoid isolates/condition analyzed in triplicate, Student’s T-test. D. Average colony size of organoids measured in pixel area ± S.D. N = 3 independent organoid isolates/condition, analyzed in triplicate, Student’s T-test . E. Western analysis of MCF10A parental and Rictor ZFN lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. F-I. MCF10A parental and Rictor ZFN cells were infected with Ad.RFP or Ad.PKC-alpha. F. Western analysis of MCF10A parental and Rictor ZFN lysates, 48 hours post-infection. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. G. GST-Pak-PBD effector pull-downs followed by western analysis for Rac performed 48 hours post-infection. Quantitation was performed using Image J software. Numbers in upper panel represent Rac-GTP levels in MCF10A-Rictor ZFN compared to MCF10A parental control. Lower numbers represent Rac-GTP levels normalized to total Rac for each sample. H. Cells were assessed for invasion through Matrigel-coated transwell filters. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in triplicate. I. Cells were assessed for Annexin V-FITC. Number of Annexin V-FITC+ per total number of cells was quantitated in Image J, Student’s T-test. N = 2 independent experiments, analyzed in triplicate. J. MCF10A parental cells were assessed for invasion through Matrigel-coated transwell filters in the presence of PKC-alpha inhibitor GO6976. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in quadruplicate.
Ad(rgd) Rfp Icre, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ad+rfp/Ad(RGD)-RFP-iCre/custom%401770%4038286179
Average 93 stars, based on 1 article reviews
ad(rgd)-rfp-icre - by Bioz Stars, 2026-09
93/100 stars
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adenovirus particles encoding enhanced red fluorescent protein (ad-rfp  (SunBio Inc)
90
SunBio Inc adenovirus particles encoding enhanced red fluorescent protein (ad-rfp
A-D. Rictor FL/FL PMECs and organoids were infected with the indicated adenoviruses. A-B. Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent <t>P-PKC-alpha</t> bands normalized to total PKC-alpha levels. C . Organoids photographed after 10 days in Matrigel culture. Average number of branches/organoid ±S.D. indicated below panels. N = 3 independent organoid isolates/condition analyzed in triplicate, Student’s T-test. D. Average colony size of organoids measured in pixel area ± S.D. N = 3 independent organoid isolates/condition, analyzed in triplicate, Student’s T-test . E. Western analysis of MCF10A parental and Rictor ZFN lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. F-I. MCF10A parental and Rictor ZFN cells were infected with Ad.RFP or Ad.PKC-alpha. F. Western analysis of MCF10A parental and Rictor ZFN lysates, 48 hours post-infection. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. G. GST-Pak-PBD effector pull-downs followed by western analysis for Rac performed 48 hours post-infection. Quantitation was performed using Image J software. Numbers in upper panel represent Rac-GTP levels in MCF10A-Rictor ZFN compared to MCF10A parental control. Lower numbers represent Rac-GTP levels normalized to total Rac for each sample. H. Cells were assessed for invasion through Matrigel-coated transwell filters. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in triplicate. I. Cells were assessed for Annexin V-FITC. Number of Annexin V-FITC+ per total number of cells was quantitated in Image J, Student’s T-test. N = 2 independent experiments, analyzed in triplicate. J. MCF10A parental cells were assessed for invasion through Matrigel-coated transwell filters in the presence of PKC-alpha inhibitor GO6976. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in quadruplicate.
Adenovirus Particles Encoding Enhanced Red Fluorescent Protein (Ad Rfp, supplied by SunBio Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ad+rfp/adenovirus+particles+encoding+enhanced+red+fluorescent+protein++ad+rfp/pm24630298-47-4-11
Average 90 stars, based on 1 article reviews
adenovirus particles encoding enhanced red fluorescent protein (ad-rfp - by Bioz Stars, 2026-09
90/100 stars
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ad-rfp-creert2  (Vector Biolabs)
N/A
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ad-h-trim27-shrna  (Vector Biolabs)
N/A
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ad-h-trim27  (Vector Biolabs)
N/A
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ad-m-trim27  (Vector Biolabs)
N/A
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ad-rfp-flpo  (Vector Biolabs)
N/A
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ad-m-trim27-shrna  (Vector Biolabs)
N/A
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Image Search Results


A-D. Rictor FL/FL PMECs and organoids were infected with the indicated adenoviruses. A-B. Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha bands normalized to total PKC-alpha levels. C . Organoids photographed after 10 days in Matrigel culture. Average number of branches/organoid ±S.D. indicated below panels. N = 3 independent organoid isolates/condition analyzed in triplicate, Student’s T-test. D. Average colony size of organoids measured in pixel area ± S.D. N = 3 independent organoid isolates/condition, analyzed in triplicate, Student’s T-test . E. Western analysis of MCF10A parental and Rictor ZFN lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. F-I. MCF10A parental and Rictor ZFN cells were infected with Ad.RFP or Ad.PKC-alpha. F. Western analysis of MCF10A parental and Rictor ZFN lysates, 48 hours post-infection. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. G. GST-Pak-PBD effector pull-downs followed by western analysis for Rac performed 48 hours post-infection. Quantitation was performed using Image J software. Numbers in upper panel represent Rac-GTP levels in MCF10A-Rictor ZFN compared to MCF10A parental control. Lower numbers represent Rac-GTP levels normalized to total Rac for each sample. H. Cells were assessed for invasion through Matrigel-coated transwell filters. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in triplicate. I. Cells were assessed for Annexin V-FITC. Number of Annexin V-FITC+ per total number of cells was quantitated in Image J, Student’s T-test. N = 2 independent experiments, analyzed in triplicate. J. MCF10A parental cells were assessed for invasion through Matrigel-coated transwell filters in the presence of PKC-alpha inhibitor GO6976. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in quadruplicate.

Journal: PLoS Genetics

Article Title: mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis

doi: 10.1371/journal.pgen.1005291

Figure Lengend Snippet: A-D. Rictor FL/FL PMECs and organoids were infected with the indicated adenoviruses. A-B. Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha bands normalized to total PKC-alpha levels. C . Organoids photographed after 10 days in Matrigel culture. Average number of branches/organoid ±S.D. indicated below panels. N = 3 independent organoid isolates/condition analyzed in triplicate, Student’s T-test. D. Average colony size of organoids measured in pixel area ± S.D. N = 3 independent organoid isolates/condition, analyzed in triplicate, Student’s T-test . E. Western analysis of MCF10A parental and Rictor ZFN lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. F-I. MCF10A parental and Rictor ZFN cells were infected with Ad.RFP or Ad.PKC-alpha. F. Western analysis of MCF10A parental and Rictor ZFN lysates, 48 hours post-infection. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. G. GST-Pak-PBD effector pull-downs followed by western analysis for Rac performed 48 hours post-infection. Quantitation was performed using Image J software. Numbers in upper panel represent Rac-GTP levels in MCF10A-Rictor ZFN compared to MCF10A parental control. Lower numbers represent Rac-GTP levels normalized to total Rac for each sample. H. Cells were assessed for invasion through Matrigel-coated transwell filters. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in triplicate. I. Cells were assessed for Annexin V-FITC. Number of Annexin V-FITC+ per total number of cells was quantitated in Image J, Student’s T-test. N = 2 independent experiments, analyzed in triplicate. J. MCF10A parental cells were assessed for invasion through Matrigel-coated transwell filters in the presence of PKC-alpha inhibitor GO6976. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in quadruplicate.

Article Snippet: PKC-alpha inhibitor GO6976 (Sigma-Aldrich, 2 nm) and adenoviral particles (Ad.RFP and Ad.PKC-alpha, Vector Biolabs) were purchased.

Techniques: Infection, Western Blot, Quantitation Assay, Software, Control, Staining

A. In situ detection of GTP-bound Rac via IF detection of GST-PBD (red; nuclei stained with DAPI, blue) on mammary gland sections from 6-week old mice. N = 6 independent fields analyzed in sections from 3 independent mammary gland sections/genotype. B . Quantitation of average percent red fluorescence (Rac-GTP) relative to blue (DAPI) on mammary gland sections from 6 week old virgin Rictor WT and Rictor MGKO mice, ± S.D., Student’s T-test. C. Cells were probed with GST-PBD (red) and counterstained with DAPI (blue). GST alone (not conjugated to PBD) was used as a negative control. GST-PBD+ fraction of total PMECs was counted and average ± S.D. shown. N = 15 fields/condition, Student’s T-test. D-I Rictor FL/FL PMECs infected with Ad.Cre or Ad.LacZ (±Ad.caRac1) and analyzed. D. Western analysis of PMEC lysates. E. Cells were probed with GST-PBD (green) and counterstained with phalloidin (red). F. Transwell invasion assays were performed. Invading cells were visualized with crystal violet and counted ±S.D. N = 6 independent isolates, analyzed in duplicate, Student’s T-test. G. PMECs were assessed by TUNEL analysis. N = 3 independent cell isolates, analyzed in duplicate. Representative images shown. Average percent TUNEL+ nuclei per total epithelial nuclei quantified. Midline values indicate average, whiskers indicate S.D., Student’s T-test. H. Rictor FL/FL organoids were infected Ad.Cre or Ad.LacZ (±Ad.caRac1) prior to embedding, and were photographed after 10 days in Matrigel culture. Right panel shows average number of branches per organoid. Each data point is the average branches per colony from individual isolates, analyzed in triplicate. Midlines are the average of all isolates, whiskers indicate S.D., Student’s T-test. N = 6. I. WT organoids were cultured ±Rac inhibitor for 10 days. Number of branches per organoid quantified. Each data point is the average branches per colony from individual isolates, analyzed in triplicate. Midline values indicate average, whiskers indicate S.D., Student’s T-test. Student’s T-test. Right panel shows average organoid size ±S.D. J-K. PMECs from Rictor FL/FL mice were tranduced with control Ad.GFP versus Ad.Cre in the presence or absence of Ad.PKC-alpha, Ad.caRac, or Ad.Akt myr . PMECs were transplanted into the cleared inguinal fat pads of 4 week old recipient female mice. Mammary glands were harvested six weeks post-transplantation and epithelial architecture and branching morphogenesis in whole-mount preparations was assessed. J. Whole-mount preparations from indicated transplanted glands. K. Quantitation of average number of branches ± S.D., Student’s T-test. Representative images shown from N = 2 independent experiments.

Journal: PLoS Genetics

Article Title: mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis

doi: 10.1371/journal.pgen.1005291

Figure Lengend Snippet: A. In situ detection of GTP-bound Rac via IF detection of GST-PBD (red; nuclei stained with DAPI, blue) on mammary gland sections from 6-week old mice. N = 6 independent fields analyzed in sections from 3 independent mammary gland sections/genotype. B . Quantitation of average percent red fluorescence (Rac-GTP) relative to blue (DAPI) on mammary gland sections from 6 week old virgin Rictor WT and Rictor MGKO mice, ± S.D., Student’s T-test. C. Cells were probed with GST-PBD (red) and counterstained with DAPI (blue). GST alone (not conjugated to PBD) was used as a negative control. GST-PBD+ fraction of total PMECs was counted and average ± S.D. shown. N = 15 fields/condition, Student’s T-test. D-I Rictor FL/FL PMECs infected with Ad.Cre or Ad.LacZ (±Ad.caRac1) and analyzed. D. Western analysis of PMEC lysates. E. Cells were probed with GST-PBD (green) and counterstained with phalloidin (red). F. Transwell invasion assays were performed. Invading cells were visualized with crystal violet and counted ±S.D. N = 6 independent isolates, analyzed in duplicate, Student’s T-test. G. PMECs were assessed by TUNEL analysis. N = 3 independent cell isolates, analyzed in duplicate. Representative images shown. Average percent TUNEL+ nuclei per total epithelial nuclei quantified. Midline values indicate average, whiskers indicate S.D., Student’s T-test. H. Rictor FL/FL organoids were infected Ad.Cre or Ad.LacZ (±Ad.caRac1) prior to embedding, and were photographed after 10 days in Matrigel culture. Right panel shows average number of branches per organoid. Each data point is the average branches per colony from individual isolates, analyzed in triplicate. Midlines are the average of all isolates, whiskers indicate S.D., Student’s T-test. N = 6. I. WT organoids were cultured ±Rac inhibitor for 10 days. Number of branches per organoid quantified. Each data point is the average branches per colony from individual isolates, analyzed in triplicate. Midline values indicate average, whiskers indicate S.D., Student’s T-test. Student’s T-test. Right panel shows average organoid size ±S.D. J-K. PMECs from Rictor FL/FL mice were tranduced with control Ad.GFP versus Ad.Cre in the presence or absence of Ad.PKC-alpha, Ad.caRac, or Ad.Akt myr . PMECs were transplanted into the cleared inguinal fat pads of 4 week old recipient female mice. Mammary glands were harvested six weeks post-transplantation and epithelial architecture and branching morphogenesis in whole-mount preparations was assessed. J. Whole-mount preparations from indicated transplanted glands. K. Quantitation of average number of branches ± S.D., Student’s T-test. Representative images shown from N = 2 independent experiments.

Article Snippet: PKC-alpha inhibitor GO6976 (Sigma-Aldrich, 2 nm) and adenoviral particles (Ad.RFP and Ad.PKC-alpha, Vector Biolabs) were purchased.

Techniques: In Situ, Staining, Quantitation Assay, Fluorescence, Negative Control, Infection, Western Blot, TUNEL Assay, Cell Culture, Control, Transplantation Assay

A-G. WT PMECs and organoids were cultured in DMSO vehicle or rapamycin. PMECs were cultured 0, 0.5, or 24 hours. Organoids were cultured 10 days in Matrigel in the presence of DMSO or rapamycin. A . Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent P-Akt, P-S6, or P-PKC-alpha bands normalized to total Akt, S6, or PKC-alpha levels. B . WT organoids photographed after 10 days in Matrigel culture. Average organoid size scored as average pixel area ± S.D., Student’s T-test, N = 6 epithelial isolates, each analyzed in triplicate. C . TUNEL analysis of PMECs. Average percent TUNEL+ nuclei per total PMEC nuclei ± S.D. is shown, Student’s T-test. N = 3 independent cell isolates, analyzed in duplicate. D. PMECs were labeled with BrdU after initial pre-treatments with rapamycin for 0.5 hours or 24 hours. Average percent BrdU+ nuclei per total nuclei ± S.D. is shown, One-way ANOVA, N = 3 independent cell isolates, analyzed in triplicate. E-F. Organoids were infected with Ad.LacZ or Ad.PKC-alpha (panel E) or Ad.caRac1 (panel F) prior to embedding in Matrigel plus rapamycin or DMSO. Average number of branches/colony is shown below each image. Average organoid size scored as average pixel area ± S.D., Student’s T-test (E) and one way ANOVA (F), N = 6 epithelial isolates, each analyzed in triplicate. G. Confluent PMEC monolayers were scratch-wounded, cultured in rapamycin or DMSO, and imaged at 24 hours. Total wounded area remaining after 24 hours was measured. Values shown are the average wound area remaining ± S.D. N = 3 independent cell isolates, analyzed in triplicate.

Journal: PLoS Genetics

Article Title: mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis

doi: 10.1371/journal.pgen.1005291

Figure Lengend Snippet: A-G. WT PMECs and organoids were cultured in DMSO vehicle or rapamycin. PMECs were cultured 0, 0.5, or 24 hours. Organoids were cultured 10 days in Matrigel in the presence of DMSO or rapamycin. A . Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent P-Akt, P-S6, or P-PKC-alpha bands normalized to total Akt, S6, or PKC-alpha levels. B . WT organoids photographed after 10 days in Matrigel culture. Average organoid size scored as average pixel area ± S.D., Student’s T-test, N = 6 epithelial isolates, each analyzed in triplicate. C . TUNEL analysis of PMECs. Average percent TUNEL+ nuclei per total PMEC nuclei ± S.D. is shown, Student’s T-test. N = 3 independent cell isolates, analyzed in duplicate. D. PMECs were labeled with BrdU after initial pre-treatments with rapamycin for 0.5 hours or 24 hours. Average percent BrdU+ nuclei per total nuclei ± S.D. is shown, One-way ANOVA, N = 3 independent cell isolates, analyzed in triplicate. E-F. Organoids were infected with Ad.LacZ or Ad.PKC-alpha (panel E) or Ad.caRac1 (panel F) prior to embedding in Matrigel plus rapamycin or DMSO. Average number of branches/colony is shown below each image. Average organoid size scored as average pixel area ± S.D., Student’s T-test (E) and one way ANOVA (F), N = 6 epithelial isolates, each analyzed in triplicate. G. Confluent PMEC monolayers were scratch-wounded, cultured in rapamycin or DMSO, and imaged at 24 hours. Total wounded area remaining after 24 hours was measured. Values shown are the average wound area remaining ± S.D. N = 3 independent cell isolates, analyzed in triplicate.

Article Snippet: PKC-alpha inhibitor GO6976 (Sigma-Aldrich, 2 nm) and adenoviral particles (Ad.RFP and Ad.PKC-alpha, Vector Biolabs) were purchased.

Techniques: Cell Culture, Western Blot, Quantitation Assay, Software, TUNEL Assay, Labeling, Infection