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  • 92
    Cell Signaling Technology Inc p s6
    mTOR inhibition with rapamycin decreases MEC survival and branching. 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, <t>P-S6,</t> 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.
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    p s6  (Abcam)
    92
    Abcam p s6
    Circulating fatty acids increase <t>p-S6</t> and p-mTOR signaling in Pten knockdown neurons . Adult animals were co-injected with the Pten knockdown lentivirus (GFP shPten; green) and the mCherry expressing control virus (mCherry control; red). Mini-osmotic pumps were implanted seven days post-injection and then seven days after implantation immunohistochemistry was used to evaluate p-S6 or p-mTOR expression (blue). (A) A slight increase in p-S6 expression (blue) was detected in Pten knockdown neurons (green) when compared to control neurons (red) in vehicle treated animals. (B) In animals treated with fatty acids, a marked increase in p-S6 staining (blue) was observed in the GFP shPten cells (green) when compared to the mCherry control cells (red only). (C) Plot of individual p-S6 intensities for each knockdown and control neuron normalized to the p-S6 intensity in controls. (D) Pten knockdown resulted in an increase in p-S6 signaling in knockdown neurons compared to controls in the vehicle control as well as the fatty acid treatment groups (mCherry control Veh = 1.0 ± 0.02, GFP shPten Veh = 1.28 ± 0.03, p
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    93
    Merck KGaA p s6
    Phosphorylation capacity of pS6K1 decreased after Raptor deficiency. (A) Detection of phosphorylation levels of pS6K1 in the mandibular first molar was performed by immunohistochemistry staining at P0 (a), P7 (b), P14 (c), and P28 (d). (B) Detection of the phosphorylation levels of pS6 in the mandibular first molar was performed at P0 (e), P7 (f), P14 (g), and P28 (h). (C,D) Detection of phosphorylated S6 in the mandibular first molar at P0. (E,F) Detection of phosphorylated 4E-BP1 in the mandibular first molar at P0. DP, dental pulp; OB, odontoblasts; D, dentin. (G) Protein expression levels of p-S6K1 and <t>p-S6</t> in WT and mutant mice. (H) The DSPP expression level decreased by the S6K1 inhibitor. (I) PF-4708671 inhibited the proliferation of dental mesenchymal cells from WT mice.
    P S6, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc p s6 240
    Progressive growth of paw hemangiosarcomas and response to rapamycin. A) Paw hemangiosarcomas grow progressively as seen by the mean (±SEM) paw diameter of largest hemangiosarcoma across all mice. B) Effect of rapamycin treatment on mean (±SEM) paw tumor diameter in Tsc1 cc DARPP Cre mice. C) Immunoblotting of total <t>S6-240,</t> phospho-S6-S240, phospho-CAD, total CAD, and Tubulin expression in both paws and kidneys from wild type and Tsc1 cc DARPP Cre vehicle and rapamycin treated mice. D) Normalized quantification of expression of phospho-S6-240 relative to total S6-240 (top) and phospho-CAD relative to total CAD (bottom) in both paws and kidneys from wild type and Tsc1 cc DARPP Cre vehicle and rapamycin treated mice. n= 5 for phospho and total-S6-240 experimental groups, n=4 for each phospho and total-CAD experimental groups. * indicates P
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    88
    Cell Signaling Technology Inc p s6 ser240 244
    Primary Keratinocytes Showed Impaired Proliferation upon  Glut1  Deletion (a) Glut1  mRNA (n=4 mice per genotype) and protein levels in the epidermis and primary keratinocytes harvested from one week old  WT, K14.Glut1 fl/wt ,  and  K14.Glut1  mice.  (b)  Immunostaining of Glut1 in skins from one-week old mice. Glut1 staining is absent in epidermis from  K14.Glut1  mice. Arrow indicates persistence of Glut1 staining in the dermis (e.g. dermal endothelial cell). Similar results obtained in 3 independent experiments.  (c)  mRNA abundance of  Glut/SGLT  family members in primary cultured  WT, Glut1 fl/wt K14Cre , and keratinocytes from  K14.Glut1  mice (n=4 mice per genotype).  (d)  2-Deoxy-D-glucose uptake in primary cultured keratinocytes obtained from one week old  WT, K14.Glut1 fl/wt ,  and  K14.Glut1  mice with or without glucose inhibitor pretreatment. Similar results obtained in uptake assays from keratinocytes from 3 independent mice.  (e)  Growth rate as assessed by Crystal Violet staining and relative cell number in primary  WT, K14.Glut1 fl/wt , and keratinocytes from  K14.Glut1  mice. Identical numbers of cells were seeded in triplicate wells in 6 well plates at day 0. For Crystal Violet staining, cells were stained 3 days after seeding and similar results were obtained in 3 keratinocytes preparations. Keratinocytes from  K14.Glut1  mice show significantly decreased growth compared to keratinoctyes from  WT  controls at day 1 and 2 after plating.  (f)  Glucose consumption and lactate production over 12 hours as measured from the media of primary cultured  WT  and  K14.Glut1  (n=3 mice per genoptye) keratinocytes.  (g)  ATP levels (luminescence) were determined in primary keratinocytes from  WT  and  K14.Glut1  (n=3 mice per genotype) and normalized to protein levels.  (h)  Western Blot analysis of the expression of p-ACC Ser79, ACC, p-S6 Ser240/244, Glut1 in primary keratinocytes obtained from  WT, Glut1 fl/wt K14Cre  and  K14.Glut1  mice. Data shown as mean±s.d.  P  values were calculated by two-tailed  t -test. Results were confirmed in at least 2 independent experiments.
    P S6 Ser240 244, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cell Signaling Technology Inc anti p s6
    Activation of cell-surface CD84 elevates PD-L1 levels through the p-AKT/mTOR pathway. ( A and B ) M210B4 cells (1 × 10 5 ) were stimulated with anti-CD84 (4 μg/ml) for 15 minutes, followed by anti-FAB cross-linking for 5 minutes. ( A ) Cells were lysed, and lysates were separated on 12% (wt/vol) SDS-PAGE and blotted with <t>anti–p-S6,</t> anti–p-AKT, anti–p-ERK, or actin. Blots are representative of 3 independent experiments. ( B ) Cells were fixed, permeabilized, and subsequently stained with anti–p-AKT and anti–p-S6 antibodies followed by a secondary anti-rabbit allophycocyanin-conjugated (APC-conjugated) antibody. Histograms are representative of 2 independent experiments. ( C – E ) Primary CLL cells were stimulated with control (IgG) or anti-CD84–activating (5 μg/ml) antibodies for 5 minutes, followed by anti-FAB cross-linking for an additional 5 minutes. Then, the cells were fixed, and p-ERK, p-AKT, and p-S6 levels were analyzed by flow cytometry ( n = 3; * P
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    Cell Signaling Technology Inc p s6 kinase
    Mean (± s.e.m. ) protein levels of p-mTOR, p-S6 kinase, ubiquitin and calpastatin in skeletal muscle from sheep fetuses on the fifth day of either saline ( n  = 6) or leptin ( n  = 6) infusion.
    P S6 Kinase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson p s6 v450
    Mean (± s.e.m. ) protein levels of p-mTOR, p-S6 kinase, ubiquitin and calpastatin in skeletal muscle from sheep fetuses on the fifth day of either saline ( n  = 6) or leptin ( n  = 6) infusion.
    P S6 V450, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc immunohistochemistry p s6
    Mean (± s.e.m. ) protein levels of p-mTOR, p-S6 kinase, ubiquitin and calpastatin in skeletal muscle from sheep fetuses on the fifth day of either saline ( n  = 6) or leptin ( n  = 6) infusion.
    Immunohistochemistry P S6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc p s6 alexa 647
    Mean (± s.e.m. ) protein levels of p-mTOR, p-S6 kinase, ubiquitin and calpastatin in skeletal muscle from sheep fetuses on the fifth day of either saline ( n  = 6) or leptin ( n  = 6) infusion.
    P S6 Alexa 647, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology p s6 ser235 236
    Mean (± s.e.m. ) protein levels of p-mTOR, p-S6 kinase, ubiquitin and calpastatin in skeletal muscle from sheep fetuses on the fifth day of either saline ( n  = 6) or leptin ( n  = 6) infusion.
    P S6 Ser235 236, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    R&D Systems p s6
    In Vitro Response to Co-inhibition of MAPK and mTOR pathways in BRAF WT Glioma Cells (A–C) Cell viability responses to AZD6244 + everolimus assessed in (A) SF188 BRAF WT , ( B ) SF8628 BRAF WT and (C) SF9427 BRAF WT . For cell viability means of six replicates with standard errors are displayed. Measurements are normalized to DMSO control. Cell viability was assessed 72 hours after treatment. Pairwise comparisons were determined by Student’s t-test; * marks p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, and **** p-value ≤ 0.0001. ( D ) Apoptotic response of SF188 BRAF WT cells to combined inhibition of AZD6244 + everolimus. Apoptosis was measured by flow cytometry. The proportions of apoptotic cells were quantified 72 hours after treatment. All data points represent averages of at least four replicates. Statistical analysis includes ANOVA and Bonferroni multiple comparisons. ( E ) Cell cycle distribution of SF188 BRAF WT cells in response to combined inhibition of AZD6244 + everolimus. Analyses were performed 24 hours after treatment and depicted are averages of four replicates. Bar graphs display distribution of cells within each cell cycle phase. Error bars indicate standard error. (F) Western blot analyses of p-Akt, p-ERK and <t>p-S6</t> in SF188 BRAF WT cells. β-actin was used as loading control. Levels of p-Akt, p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above results, in samples receiving combination therapy, inhibitors were administered simultaneously.
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    Santa Cruz Biotechnology p s6
    mTORC1 positively regulates OPN expression. (A) Tca8113 and KB cells were treated with or without 50 nM rapamycin (Rapa) for 24 h. Expression levels of OPN, <t>p-S6</t> and S6 were detected by western blot analysis (top). The mRNA levels of OPN were analyzed by qRT-PCR (middle). OPN levels in the cell supernatants were detected by an ELISA (bottom). Data indicate mean ± SD of triplicate samples. ** P
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    Oncothyreon p s6
    mTORC1 positively regulates OPN expression. (A) Tca8113 and KB cells were treated with or without 50 nM rapamycin (Rapa) for 24 h. Expression levels of OPN, <t>p-S6</t> and S6 were detected by western blot analysis (top). The mRNA levels of OPN were analyzed by qRT-PCR (middle). OPN levels in the cell supernatants were detected by an ELISA (bottom). Data indicate mean ± SD of triplicate samples. ** P
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    Millipore p s6
    RAPA inhibited the over-activation mTOR pathway to revers the senescence of MSCs from MRL/lpr mice ( A ) The over-expression of p-mTOR, p-S6K and <t>p-S6</t> in p4 MSCs from MRL/lpr mice compared with normal group were determined by western blot analysis. The treatment of RAPA could obviously inhibit the expression of those proteins. GAPDH was used as the internal control. ( B ) P4 MSCs cultured into 24-well plates. The expression of p-mTOR, p-S6K and p-S6 analyzed by immunofluorescence staining showed that their over-activation in MSCs from MRL/lpr mice could be inhibited by RAPA treatment. Counterstaining with DAPI displays the localization of the nucleus (Scale bar = 50 μm). All data were expressed as the mean±SEM (n = 3,*P
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    Cell Signaling Technology Inc antibody against p s6
    RAPA inhibited the over-activation mTOR pathway to revers the senescence of MSCs from MRL/lpr mice ( A ) The over-expression of p-mTOR, p-S6K and <t>p-S6</t> in p4 MSCs from MRL/lpr mice compared with normal group were determined by western blot analysis. The treatment of RAPA could obviously inhibit the expression of those proteins. GAPDH was used as the internal control. ( B ) P4 MSCs cultured into 24-well plates. The expression of p-mTOR, p-S6K and p-S6 analyzed by immunofluorescence staining showed that their over-activation in MSCs from MRL/lpr mice could be inhibited by RAPA treatment. Counterstaining with DAPI displays the localization of the nucleus (Scale bar = 50 μm). All data were expressed as the mean±SEM (n = 3,*P
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    Cell Signaling Technology Inc anti p s6 ax647
    RAPA inhibited the over-activation mTOR pathway to revers the senescence of MSCs from MRL/lpr mice ( A ) The over-expression of p-mTOR, p-S6K and <t>p-S6</t> in p4 MSCs from MRL/lpr mice compared with normal group were determined by western blot analysis. The treatment of RAPA could obviously inhibit the expression of those proteins. GAPDH was used as the internal control. ( B ) P4 MSCs cultured into 24-well plates. The expression of p-mTOR, p-S6K and p-S6 analyzed by immunofluorescence staining showed that their over-activation in MSCs from MRL/lpr mice could be inhibited by RAPA treatment. Counterstaining with DAPI displays the localization of the nucleus (Scale bar = 50 μm). All data were expressed as the mean±SEM (n = 3,*P
    Anti P S6 Ax647, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p s6 ribosomal
    RAPA inhibited the over-activation mTOR pathway to revers the senescence of MSCs from MRL/lpr mice ( A ) The over-expression of p-mTOR, p-S6K and <t>p-S6</t> in p4 MSCs from MRL/lpr mice compared with normal group were determined by western blot analysis. The treatment of RAPA could obviously inhibit the expression of those proteins. GAPDH was used as the internal control. ( B ) P4 MSCs cultured into 24-well plates. The expression of p-mTOR, p-S6K and p-S6 analyzed by immunofluorescence staining showed that their over-activation in MSCs from MRL/lpr mice could be inhibited by RAPA treatment. Counterstaining with DAPI displays the localization of the nucleus (Scale bar = 50 μm). All data were expressed as the mean±SEM (n = 3,*P
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    Image Search Results


    mTOR inhibition with rapamycin decreases MEC survival and branching. 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: mTOR inhibition with rapamycin decreases MEC survival and branching. 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: IHC on paraffin-embedded sections was performed as described previously [ ] using: Ki67 (Santa Cruz Biotechnologies), P-S6 (Cell Signaling Technologies); P-Akt S473 (Cell Signaling Technologies); Rictor (Santa Cruz), E-cadherin (Transduction Labs).

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

    Impaired survival and morphogenesis of mammary epithelial structures upon loss of Rictor ex vivo . A. Western analysis of whole mammary lysates harvested from 10-week old female mice. B-C. Rictor FL/FL PMECs were infected with Ad.Cre or Ad.LacZ and cultured 7 days. B. Western analysis of PMEC lysates under serum starved conditions. Quantitation was performed using Image J software and numbers represent P-Akt or P-S6 bands normalized to total Akt or S6 levels. C. BrdU+ (left) and TUNEL+ (right) nuclei relative to total nuclei were quantified. N = 5 epithelial isolates, each analyzed in triplicate. Midline values indicate average, whiskers indicate S.D., Student’s T-test. D-E. MCF10A and MCF10A-Rictor ZFN cells were analyzed. D. Western analysis of MCF10A lysates. E. Cells were labeled with Annexin V-FITC for 6 hours then photographed. Percent Annexin V+ cells versus total was quantified, average ± S.D. shown. N = 3 independent experiments, each analyzed in quadruplicate, representative images shown, Student’s T-test. F-I. Rictor FL/FL PMECs and organoids were infected with Ad.Cre or Ad.LacZ. F. Organoids photographed after 10 days in Matrigel culture. Representative images are shown. G. Average organoid size, measured in pixel area using Image J software, ± S.D. (left panel) and average number of branches/organoid ± S.D. (right panel) are shown. N = 6 independent organoid isolates, analyzed in triplicate, Student’s T-test. H. Western analysis of PMEC lysates. I. Transwell invasion of adenovirus-infected Rictor FL/FL PMECs in response to serum. Cells migrating to the opposite side of the transwell filter were visualized with crystal violet and counted. N = 6 PMEC isolates/condition, one-way ANOVA. J. Transwell invasion of MCF10A and MCF10A-Rictor ZFN cells in response to serum is shown as average number of invading cells ± S.D. N = 3, independent experiments, each analyzed in triplicate, Student’s T-test.

    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: Impaired survival and morphogenesis of mammary epithelial structures upon loss of Rictor ex vivo . A. Western analysis of whole mammary lysates harvested from 10-week old female mice. B-C. Rictor FL/FL PMECs were infected with Ad.Cre or Ad.LacZ and cultured 7 days. B. Western analysis of PMEC lysates under serum starved conditions. Quantitation was performed using Image J software and numbers represent P-Akt or P-S6 bands normalized to total Akt or S6 levels. C. BrdU+ (left) and TUNEL+ (right) nuclei relative to total nuclei were quantified. N = 5 epithelial isolates, each analyzed in triplicate. Midline values indicate average, whiskers indicate S.D., Student’s T-test. D-E. MCF10A and MCF10A-Rictor ZFN cells were analyzed. D. Western analysis of MCF10A lysates. E. Cells were labeled with Annexin V-FITC for 6 hours then photographed. Percent Annexin V+ cells versus total was quantified, average ± S.D. shown. N = 3 independent experiments, each analyzed in quadruplicate, representative images shown, Student’s T-test. F-I. Rictor FL/FL PMECs and organoids were infected with Ad.Cre or Ad.LacZ. F. Organoids photographed after 10 days in Matrigel culture. Representative images are shown. G. Average organoid size, measured in pixel area using Image J software, ± S.D. (left panel) and average number of branches/organoid ± S.D. (right panel) are shown. N = 6 independent organoid isolates, analyzed in triplicate, Student’s T-test. H. Western analysis of PMEC lysates. I. Transwell invasion of adenovirus-infected Rictor FL/FL PMECs in response to serum. Cells migrating to the opposite side of the transwell filter were visualized with crystal violet and counted. N = 6 PMEC isolates/condition, one-way ANOVA. J. Transwell invasion of MCF10A and MCF10A-Rictor ZFN cells in response to serum is shown as average number of invading cells ± S.D. N = 3, independent experiments, each analyzed in triplicate, Student’s T-test.

    Article Snippet: IHC on paraffin-embedded sections was performed as described previously [ ] using: Ki67 (Santa Cruz Biotechnologies), P-S6 (Cell Signaling Technologies); P-Akt S473 (Cell Signaling Technologies); Rictor (Santa Cruz), E-cadherin (Transduction Labs).

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

    Unlike mTORC2, mTORC1 is dispensable for MEC survival and branching morphogenesis. A-C. Raptor FL/FL PMECs and organoids were infected with Ad.Cre and Ad.LacZ, and cultured 10 days. A. Western analysis of PMECs cultured in the absence of serum. Quantitation was performed using Image J software and numbers represent P-Akt or P-S6 bands normalized to total Akt or S6 levels. B. Organoids were infected with Ad.Cre or Ad.LacZ photographed after 10 days in Matrigel culture. Representative images are shown. Average number of branches/organoid for each group is shown below the image. Average organoid size (pixels) ± S.D. is shown, Student’s T-test. N = 6 independent organoid isolates, analyzed in triplicate . C. PMECs from Raptor Fl/FL mice were infected with Ad.LacZ or Ad.Cre, grown to confluence and scratch-wounded. Monolayers were imaged. Total wound area remaining was measured 24 hours after wounding. Values shown are the avg ± S.D. N = 6 per time point, Student’s T-test. D-E. Mammary glands from virgin female Raptor WT and Raptor MGKO at 6 and 10 weeks of age were analyzed. N = 10 mice per genotype at each time point. Statistical analysis performed with Student’s T-test. D. IHC for Raptor, P-S6, Ki67+, and TUNEL+ nuclei in mammary glands of 6-week old mice. Representative images are shown. Scale bars = 50 microns. Average percent Ki67+ nuclei and TUNEL+ nuclei (± S.D) per total epithelial nuclei was determined E. Whole mount hematoxylin staining of mammary glands. Representative images are shown. LN = lymph node. The ductal length beyond the mammary lymph node was measured in whole mounted mammary glands. Average length (in microns) ± S.D. is shown. The number of T-shaped side branches was enumerated in whole mounted mammary glands. Values shown represent average number of side branches ± S.D.

    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: Unlike mTORC2, mTORC1 is dispensable for MEC survival and branching morphogenesis. A-C. Raptor FL/FL PMECs and organoids were infected with Ad.Cre and Ad.LacZ, and cultured 10 days. A. Western analysis of PMECs cultured in the absence of serum. Quantitation was performed using Image J software and numbers represent P-Akt or P-S6 bands normalized to total Akt or S6 levels. B. Organoids were infected with Ad.Cre or Ad.LacZ photographed after 10 days in Matrigel culture. Representative images are shown. Average number of branches/organoid for each group is shown below the image. Average organoid size (pixels) ± S.D. is shown, Student’s T-test. N = 6 independent organoid isolates, analyzed in triplicate . C. PMECs from Raptor Fl/FL mice were infected with Ad.LacZ or Ad.Cre, grown to confluence and scratch-wounded. Monolayers were imaged. Total wound area remaining was measured 24 hours after wounding. Values shown are the avg ± S.D. N = 6 per time point, Student’s T-test. D-E. Mammary glands from virgin female Raptor WT and Raptor MGKO at 6 and 10 weeks of age were analyzed. N = 10 mice per genotype at each time point. Statistical analysis performed with Student’s T-test. D. IHC for Raptor, P-S6, Ki67+, and TUNEL+ nuclei in mammary glands of 6-week old mice. Representative images are shown. Scale bars = 50 microns. Average percent Ki67+ nuclei and TUNEL+ nuclei (± S.D) per total epithelial nuclei was determined E. Whole mount hematoxylin staining of mammary glands. Representative images are shown. LN = lymph node. The ductal length beyond the mammary lymph node was measured in whole mounted mammary glands. Average length (in microns) ± S.D. is shown. The number of T-shaped side branches was enumerated in whole mounted mammary glands. Values shown represent average number of side branches ± S.D.

    Article Snippet: IHC on paraffin-embedded sections was performed as described previously [ ] using: Ki67 (Santa Cruz Biotechnologies), P-S6 (Cell Signaling Technologies); P-Akt S473 (Cell Signaling Technologies); Rictor (Santa Cruz), E-cadherin (Transduction Labs).

    Techniques: Infection, Cell Culture, Western Blot, Quantitation Assay, Software, Mouse Assay, Immunohistochemistry, TUNEL Assay, Staining

    Westerns blots of frontal cortex of  Fmr1  KO and control mice on vehicle and rapamycin treatment.  (A)  Representative Western blot images.  (B)  p-mTOR levels did not differ among the groups.  (C)  mTOR levels did not differ among the groups.  (D)  p-mTOR/Total mTOR did not differ among the groups.  (E)  p-p70S6k did not differ among the groups.  (F)  p70S6k did not differ among the groups.  (G)  p-p70S6k/Total p70S6k did not differ among the groups.  (H)  The genotype × treatment interaction for pS6 235/236 was statistically significant.  Post hoc t -tests revealed that vehicle-treated  Fmr1  KO animals had significantly higher p-S6 235/236 ( p  = 0.002) compared to vehicle-treated controls. This was significantly reduced by rapamycin treatment ( p  = 0.002).  (I)  S6 levels did not differ among groups.  (J)  The genotype × treatment interaction for p-S6 (235/236)/Total S6 approached statistical significance. We looked at individual differences by means of  post hoc t -tests and found that the difference between vehicle-treated controls and vehicle-treated  Fmr1  KO mice was statistically significant ( p  = 0.004).  (K)  The genotype × treatment interaction for p-S6 240/244 was statistically significant. ( Post hoc t -tests revealed that vehicle-treated  Fmr1  KO animals had significantly higher p-S6 240/244 levels compared to vehicle-treated controls ( p  = 0.010). This was significantly reduced with rapamycin treatment ( p  = 0.006).  (L)  Total S6 levels did not differ among the groups.  (M)  The genotype × treatment interaction for p-S6 (240/244)/Total S6 approached statistical significance. We looked at individual differences by means of  post hoc t -tests and found that the difference between vehicle-treated controls and vehicle-treated  Fmr1  KO mice was statistically significant ( p  = 0.016).  (N)  The genotype × treatment interaction for p-AKT Ser473 was statistically significant.  Post hoc t -tests revealed that vehicle-treated  Fmr1  KO animals had higher p-AKT compared to vehicle-treated controls ( p  = 0.020). p-AKT Ser473 levels were reduced in  Fmr1  KO animals after rapamycin treatment ( p  = 0.013).  (O)  Total AKT levels did not differ among the groups.  (P)  p-Akt (473)/Akt did not differ among the groups.  (Q)  The main effect of treatment for p-ERK levels was statistically significant indicating that regardless of genotype, rapamycin reduced p-ERK.  (R)  The main effect of treatment for ERK levels was statistically significant indicating that regardless of genotype, rapamycin reduced ERK.  (S)  p-ERK/ERK did not differ among the groups.  (B–S)  Levels were normalized to total protein in the blot. Values presented are relative to the mean of vehicle-treated control values. Bars represent mean ± SEM. ∼0.05

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Negative Effects of Chronic Rapamycin Treatment on Behavior in a Mouse Model of Fragile X Syndrome

    doi: 10.3389/fnmol.2017.00452

    Figure Lengend Snippet: Westerns blots of frontal cortex of Fmr1 KO and control mice on vehicle and rapamycin treatment. (A) Representative Western blot images. (B) p-mTOR levels did not differ among the groups. (C) mTOR levels did not differ among the groups. (D) p-mTOR/Total mTOR did not differ among the groups. (E) p-p70S6k did not differ among the groups. (F) p70S6k did not differ among the groups. (G) p-p70S6k/Total p70S6k did not differ among the groups. (H) The genotype × treatment interaction for pS6 235/236 was statistically significant. Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had significantly higher p-S6 235/236 ( p = 0.002) compared to vehicle-treated controls. This was significantly reduced by rapamycin treatment ( p = 0.002). (I) S6 levels did not differ among groups. (J) The genotype × treatment interaction for p-S6 (235/236)/Total S6 approached statistical significance. We looked at individual differences by means of post hoc t -tests and found that the difference between vehicle-treated controls and vehicle-treated Fmr1 KO mice was statistically significant ( p = 0.004). (K) The genotype × treatment interaction for p-S6 240/244 was statistically significant. ( Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had significantly higher p-S6 240/244 levels compared to vehicle-treated controls ( p = 0.010). This was significantly reduced with rapamycin treatment ( p = 0.006). (L) Total S6 levels did not differ among the groups. (M) The genotype × treatment interaction for p-S6 (240/244)/Total S6 approached statistical significance. We looked at individual differences by means of post hoc t -tests and found that the difference between vehicle-treated controls and vehicle-treated Fmr1 KO mice was statistically significant ( p = 0.016). (N) The genotype × treatment interaction for p-AKT Ser473 was statistically significant. Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had higher p-AKT compared to vehicle-treated controls ( p = 0.020). p-AKT Ser473 levels were reduced in Fmr1 KO animals after rapamycin treatment ( p = 0.013). (O) Total AKT levels did not differ among the groups. (P) p-Akt (473)/Akt did not differ among the groups. (Q) The main effect of treatment for p-ERK levels was statistically significant indicating that regardless of genotype, rapamycin reduced p-ERK. (R) The main effect of treatment for ERK levels was statistically significant indicating that regardless of genotype, rapamycin reduced ERK. (S) p-ERK/ERK did not differ among the groups. (B–S) Levels were normalized to total protein in the blot. Values presented are relative to the mean of vehicle-treated control values. Bars represent mean ± SEM. ∼0.05

    Article Snippet: Primary antibodies were used at a 1:1000 dilution and were as follows: FMRP (Abcam 27455), p-mTOR (Cell Signaling 5536), mTOR (Cell Signaling 2983), p-p70S6K (Cell Signaling 9234), p70S6K (Cell Signaling 2708), p-S6 235/236 (Cell Signaling 2211), p-S6 240/244 (Cell Signaling 2215), S6 (Cell Signaling 2217), p-ERK (Cell Signaling 4370), ERK (Cell Signaling 7124), p-AKT Ser473 (Cell Signaling 4060), and AKT (Cell Signaling 9272).

    Techniques: Mouse Assay, Western Blot

    Effects of NVP-BEZ235 on cell viability and PI3K/mTOR signaling are independent of PIK3CA status. (A) Cell viability of mutant and wild-type isogenic PIK3CA cells was assessed by MTS assay after treatment with increasing concentrations (0–500 nM) of NVP-BEZ235 for 48 hours. Results shown are the mean of four independent experiments. (B) Western blot analysis for p-AKT Thr308 , p-AKT Ser473 , p-S6 Ser240/244 , p-S6 Ser235/236 , cleaved caspase 3, and cleaved PARP was performed after 2, 6, 24, and 48 hours incubation with (−) 0 or (+) 500 nM NVP-BEZ235.

    Journal: PLoS ONE

    Article Title: The Dual PI3K/mTOR Inhibitor NVP-BEZ235 Induces Tumor Regression in a Genetically Engineered Mouse Model of PIK3CA Wild-Type Colorectal Cancer

    doi: 10.1371/journal.pone.0025132

    Figure Lengend Snippet: Effects of NVP-BEZ235 on cell viability and PI3K/mTOR signaling are independent of PIK3CA status. (A) Cell viability of mutant and wild-type isogenic PIK3CA cells was assessed by MTS assay after treatment with increasing concentrations (0–500 nM) of NVP-BEZ235 for 48 hours. Results shown are the mean of four independent experiments. (B) Western blot analysis for p-AKT Thr308 , p-AKT Ser473 , p-S6 Ser240/244 , p-S6 Ser235/236 , cleaved caspase 3, and cleaved PARP was performed after 2, 6, 24, and 48 hours incubation with (−) 0 or (+) 500 nM NVP-BEZ235.

    Article Snippet: Detection was performed using the Amersham™ ECL™ Western Blot Detection Reagents (GE Healthcare). p-AKT Thr308 (1∶1000 dilution), p-AKT Ser473 (1∶2000 dilution), total AKT (1∶1000 dilution), p-S6 Ser240/244 (1∶3000 dilution), p-S6 Ser235/236 (1∶1000 dilution), S6 (1∶1000 dilution), cleaved caspase 3 (1∶1000 dilution), and cleaved PARP (1∶1000 dilution) were obtained from Cell Signaling Technologies (Beverly, MA). β-actin (1∶5000 dilution) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Mutagenesis, MTS Assay, Western Blot, Incubation

    In vivo NVP-BEZ235 treatment of a GEM model for sporadic CRC results in transient PI3K blockade inhibition and sustained mTORC1/mTORC2 blockade. Mice with colonic tumors were randomized to treatment with control diluent or 45 mg/kg NVP-BEZ235 by daily oral gavage for five days. Western blot analysis of p-AKT Thr308 , p-AKT Ser473 , and p-S6 Ser240/244 was performed for tumors treated with (−) control diluent or (+) NVP-BEZ235 for (A) five and (B) 28 days. (C) Immunohistochemistry of p-AKT Ser473 , p-S6 Ser240/244 , and p-S6 Ser235/236 was performed for tumors treated with control diluent or NVP-BEZ235 for 28 days.

    Journal: PLoS ONE

    Article Title: The Dual PI3K/mTOR Inhibitor NVP-BEZ235 Induces Tumor Regression in a Genetically Engineered Mouse Model of PIK3CA Wild-Type Colorectal Cancer

    doi: 10.1371/journal.pone.0025132

    Figure Lengend Snippet: In vivo NVP-BEZ235 treatment of a GEM model for sporadic CRC results in transient PI3K blockade inhibition and sustained mTORC1/mTORC2 blockade. Mice with colonic tumors were randomized to treatment with control diluent or 45 mg/kg NVP-BEZ235 by daily oral gavage for five days. Western blot analysis of p-AKT Thr308 , p-AKT Ser473 , and p-S6 Ser240/244 was performed for tumors treated with (−) control diluent or (+) NVP-BEZ235 for (A) five and (B) 28 days. (C) Immunohistochemistry of p-AKT Ser473 , p-S6 Ser240/244 , and p-S6 Ser235/236 was performed for tumors treated with control diluent or NVP-BEZ235 for 28 days.

    Article Snippet: Detection was performed using the Amersham™ ECL™ Western Blot Detection Reagents (GE Healthcare). p-AKT Thr308 (1∶1000 dilution), p-AKT Ser473 (1∶2000 dilution), total AKT (1∶1000 dilution), p-S6 Ser240/244 (1∶3000 dilution), p-S6 Ser235/236 (1∶1000 dilution), S6 (1∶1000 dilution), cleaved caspase 3 (1∶1000 dilution), and cleaved PARP (1∶1000 dilution) were obtained from Cell Signaling Technologies (Beverly, MA). β-actin (1∶5000 dilution) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: In Vivo, Inhibition, Mouse Assay, Western Blot, Immunohistochemistry

    NVP-BEZ235 treatment results in decreased cell viability, transient PI3K blockade inhibition, and sustained mTORC1/mTORC2 blockade. (A) Cell viability of HCT116, DLD-1, and SW480 CRC cell lines was assessed by MTS assay after treatment with increasing concentrations (0–500 nM) of NVP-BEZ235 for 48 hours. Results shown are the mean of three independent experiments. (B) Western blot analysis for p-AKT Thr308 , p-AKT Ser473 , p-S6 Ser240/244 , p-S6 Ser235/236 , cleaved caspase 3, and cleaved PARP was performed after 2, 6, 24, and 48 hours incubation with (−) 0 or (+) 500 nM NVP-BEZ235.

    Journal: PLoS ONE

    Article Title: The Dual PI3K/mTOR Inhibitor NVP-BEZ235 Induces Tumor Regression in a Genetically Engineered Mouse Model of PIK3CA Wild-Type Colorectal Cancer

    doi: 10.1371/journal.pone.0025132

    Figure Lengend Snippet: NVP-BEZ235 treatment results in decreased cell viability, transient PI3K blockade inhibition, and sustained mTORC1/mTORC2 blockade. (A) Cell viability of HCT116, DLD-1, and SW480 CRC cell lines was assessed by MTS assay after treatment with increasing concentrations (0–500 nM) of NVP-BEZ235 for 48 hours. Results shown are the mean of three independent experiments. (B) Western blot analysis for p-AKT Thr308 , p-AKT Ser473 , p-S6 Ser240/244 , p-S6 Ser235/236 , cleaved caspase 3, and cleaved PARP was performed after 2, 6, 24, and 48 hours incubation with (−) 0 or (+) 500 nM NVP-BEZ235.

    Article Snippet: Detection was performed using the Amersham™ ECL™ Western Blot Detection Reagents (GE Healthcare). p-AKT Thr308 (1∶1000 dilution), p-AKT Ser473 (1∶2000 dilution), total AKT (1∶1000 dilution), p-S6 Ser240/244 (1∶3000 dilution), p-S6 Ser235/236 (1∶1000 dilution), S6 (1∶1000 dilution), cleaved caspase 3 (1∶1000 dilution), and cleaved PARP (1∶1000 dilution) were obtained from Cell Signaling Technologies (Beverly, MA). β-actin (1∶5000 dilution) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Inhibition, MTS Assay, Western Blot, Incubation

    Circulating fatty acids increase p-S6 and p-mTOR signaling in Pten knockdown neurons . Adult animals were co-injected with the Pten knockdown lentivirus (GFP shPten; green) and the mCherry expressing control virus (mCherry control; red). Mini-osmotic pumps were implanted seven days post-injection and then seven days after implantation immunohistochemistry was used to evaluate p-S6 or p-mTOR expression (blue). (A) A slight increase in p-S6 expression (blue) was detected in Pten knockdown neurons (green) when compared to control neurons (red) in vehicle treated animals. (B) In animals treated with fatty acids, a marked increase in p-S6 staining (blue) was observed in the GFP shPten cells (green) when compared to the mCherry control cells (red only). (C) Plot of individual p-S6 intensities for each knockdown and control neuron normalized to the p-S6 intensity in controls. (D) Pten knockdown resulted in an increase in p-S6 signaling in knockdown neurons compared to controls in the vehicle control as well as the fatty acid treatment groups (mCherry control Veh = 1.0 ± 0.02, GFP shPten Veh = 1.28 ± 0.03, p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Fatty acids increase neuronal hypertrophy of Pten knockdown neurons

    doi: 10.3389/fnmol.2014.00030

    Figure Lengend Snippet: Circulating fatty acids increase p-S6 and p-mTOR signaling in Pten knockdown neurons . Adult animals were co-injected with the Pten knockdown lentivirus (GFP shPten; green) and the mCherry expressing control virus (mCherry control; red). Mini-osmotic pumps were implanted seven days post-injection and then seven days after implantation immunohistochemistry was used to evaluate p-S6 or p-mTOR expression (blue). (A) A slight increase in p-S6 expression (blue) was detected in Pten knockdown neurons (green) when compared to control neurons (red) in vehicle treated animals. (B) In animals treated with fatty acids, a marked increase in p-S6 staining (blue) was observed in the GFP shPten cells (green) when compared to the mCherry control cells (red only). (C) Plot of individual p-S6 intensities for each knockdown and control neuron normalized to the p-S6 intensity in controls. (D) Pten knockdown resulted in an increase in p-S6 signaling in knockdown neurons compared to controls in the vehicle control as well as the fatty acid treatment groups (mCherry control Veh = 1.0 ± 0.02, GFP shPten Veh = 1.28 ± 0.03, p

    Article Snippet: For p-S6 (Ser235/236), p-mTOR (Ser2448) and Pten staining, the sections were stained with chicken anti-GFP (1:1000, abcam), mouse anti-mCherry, and rabbit anti-p-S6 (1:100, Cell Signaling) or rabbit anti-Pten (1:100, Cell Signaling) or rabbit anti-p-mTOR (1:50, Cell Signaling) and incubated overnight at 4°C.

    Techniques: Injection, Expressing, Immunohistochemistry, Staining

    p-S6/p-mTOR correlated to increased soma size in Pten knockdown neurons . Adult animals were co-injected with the Pten knockdown lentivirus (GFP shPten; green) and the mCherry expressing control virus (mCherry control; red) and immunohistochemistry was used to evaluate p-S6 or p-mTOR expression (blue) at 21 days post-injection. (A,B) Increased p-S6 or p-mTOR staining (blue) was observed in the GFP shPten cells (green) when compared to neighboring mCherry control cells (red) in the same histological section. (C) The fluorescence intensity of the p-S6 staining was measured and normalized to the average value obtained for control cells in each histological section. A linear regression analysis was then used to demonstrate that there is a positive correlation between p-S6 staining intensity and soma size. A best fit line for the Pten knockdown neurons (solid) is shown with the 95% confidence interval (dashed) (slope = 0.018 ± 0.003; R 2 = 0.3734, p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Fatty acids increase neuronal hypertrophy of Pten knockdown neurons

    doi: 10.3389/fnmol.2014.00030

    Figure Lengend Snippet: p-S6/p-mTOR correlated to increased soma size in Pten knockdown neurons . Adult animals were co-injected with the Pten knockdown lentivirus (GFP shPten; green) and the mCherry expressing control virus (mCherry control; red) and immunohistochemistry was used to evaluate p-S6 or p-mTOR expression (blue) at 21 days post-injection. (A,B) Increased p-S6 or p-mTOR staining (blue) was observed in the GFP shPten cells (green) when compared to neighboring mCherry control cells (red) in the same histological section. (C) The fluorescence intensity of the p-S6 staining was measured and normalized to the average value obtained for control cells in each histological section. A linear regression analysis was then used to demonstrate that there is a positive correlation between p-S6 staining intensity and soma size. A best fit line for the Pten knockdown neurons (solid) is shown with the 95% confidence interval (dashed) (slope = 0.018 ± 0.003; R 2 = 0.3734, p

    Article Snippet: For p-S6 (Ser235/236), p-mTOR (Ser2448) and Pten staining, the sections were stained with chicken anti-GFP (1:1000, abcam), mouse anti-mCherry, and rabbit anti-p-S6 (1:100, Cell Signaling) or rabbit anti-Pten (1:100, Cell Signaling) or rabbit anti-p-mTOR (1:50, Cell Signaling) and incubated overnight at 4°C.

    Techniques: Injection, Expressing, Immunohistochemistry, Staining, Fluorescence

    Rapamycin Inhibits Neuronal Hypertrophy and Increased p-S6 signaling Caused by Pten Knockdown (A) Pten knockdown neurons treated with fatty acids (green) have an increase in soma size and p-S6 intensity (blue) compared to neighboring control neurons (red). (B) Treating these fatty acid exposed, Pten knockdown neurons with rapamycin (green) inhibits this increase in soma size and increase in p-S6 staining (blue) compared to control neurons (red). (C) In vehicle treated neurons, there is still an increase in soma size when comparing the knockdown neurons to control neurons (Vehicle control = 94.13 ± 1.36, Vehicle shPten = 107.4 ± 1.52, p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Fatty acids increase neuronal hypertrophy of Pten knockdown neurons

    doi: 10.3389/fnmol.2014.00030

    Figure Lengend Snippet: Rapamycin Inhibits Neuronal Hypertrophy and Increased p-S6 signaling Caused by Pten Knockdown (A) Pten knockdown neurons treated with fatty acids (green) have an increase in soma size and p-S6 intensity (blue) compared to neighboring control neurons (red). (B) Treating these fatty acid exposed, Pten knockdown neurons with rapamycin (green) inhibits this increase in soma size and increase in p-S6 staining (blue) compared to control neurons (red). (C) In vehicle treated neurons, there is still an increase in soma size when comparing the knockdown neurons to control neurons (Vehicle control = 94.13 ± 1.36, Vehicle shPten = 107.4 ± 1.52, p

    Article Snippet: For p-S6 (Ser235/236), p-mTOR (Ser2448) and Pten staining, the sections were stained with chicken anti-GFP (1:1000, abcam), mouse anti-mCherry, and rabbit anti-p-S6 (1:100, Cell Signaling) or rabbit anti-Pten (1:100, Cell Signaling) or rabbit anti-p-mTOR (1:50, Cell Signaling) and incubated overnight at 4°C.

    Techniques: Staining

    Phosphorylation capacity of pS6K1 decreased after Raptor deficiency. (A) Detection of phosphorylation levels of pS6K1 in the mandibular first molar was performed by immunohistochemistry staining at P0 (a), P7 (b), P14 (c), and P28 (d). (B) Detection of the phosphorylation levels of pS6 in the mandibular first molar was performed at P0 (e), P7 (f), P14 (g), and P28 (h). (C,D) Detection of phosphorylated S6 in the mandibular first molar at P0. (E,F) Detection of phosphorylated 4E-BP1 in the mandibular first molar at P0. DP, dental pulp; OB, odontoblasts; D, dentin. (G) Protein expression levels of p-S6K1 and p-S6 in WT and mutant mice. (H) The DSPP expression level decreased by the S6K1 inhibitor. (I) PF-4708671 inhibited the proliferation of dental mesenchymal cells from WT mice.

    Journal: Frontiers in Physiology

    Article Title: Conditional Knockout of Raptor/mTORC1 Results in Dentin Malformation

    doi: 10.3389/fphys.2019.00250

    Figure Lengend Snippet: Phosphorylation capacity of pS6K1 decreased after Raptor deficiency. (A) Detection of phosphorylation levels of pS6K1 in the mandibular first molar was performed by immunohistochemistry staining at P0 (a), P7 (b), P14 (c), and P28 (d). (B) Detection of the phosphorylation levels of pS6 in the mandibular first molar was performed at P0 (e), P7 (f), P14 (g), and P28 (h). (C,D) Detection of phosphorylated S6 in the mandibular first molar at P0. (E,F) Detection of phosphorylated 4E-BP1 in the mandibular first molar at P0. DP, dental pulp; OB, odontoblasts; D, dentin. (G) Protein expression levels of p-S6K1 and p-S6 in WT and mutant mice. (H) The DSPP expression level decreased by the S6K1 inhibitor. (I) PF-4708671 inhibited the proliferation of dental mesenchymal cells from WT mice.

    Article Snippet: Primary antibodies used in the experiments included mouse polyclonal anti-Ki67 antibody (gift from Prof. Tong), Raptor, mTOR (1:200, Lifespan BioSciences, Inc., Seattle, USA), p-S6K1, p-S6 (1:200, Merck Millipore, Darmstadt, Germany) Dspp, and OCN (Santa Cruz Biotechnology, Inc., California, USA).

    Techniques: Immunohistochemistry, Staining, Expressing, Mutagenesis, Mouse Assay

    Progressive growth of paw hemangiosarcomas and response to rapamycin. A) Paw hemangiosarcomas grow progressively as seen by the mean (±SEM) paw diameter of largest hemangiosarcoma across all mice. B) Effect of rapamycin treatment on mean (±SEM) paw tumor diameter in Tsc1 cc DARPP Cre mice. C) Immunoblotting of total S6-240, phospho-S6-S240, phospho-CAD, total CAD, and Tubulin expression in both paws and kidneys from wild type and Tsc1 cc DARPP Cre vehicle and rapamycin treated mice. D) Normalized quantification of expression of phospho-S6-240 relative to total S6-240 (top) and phospho-CAD relative to total CAD (bottom) in both paws and kidneys from wild type and Tsc1 cc DARPP Cre vehicle and rapamycin treated mice. n= 5 for phospho and total-S6-240 experimental groups, n=4 for each phospho and total-CAD experimental groups. * indicates P

    Journal: Molecular cancer research : MCR

    Article Title: A Vascular Model of Tsc1 Deficiency Accelerates Renal Tumor Formation with Accompanying Hemangiosarcomas

    doi: 10.1158/1541-7786.MCR-14-0178

    Figure Lengend Snippet: Progressive growth of paw hemangiosarcomas and response to rapamycin. A) Paw hemangiosarcomas grow progressively as seen by the mean (±SEM) paw diameter of largest hemangiosarcoma across all mice. B) Effect of rapamycin treatment on mean (±SEM) paw tumor diameter in Tsc1 cc DARPP Cre mice. C) Immunoblotting of total S6-240, phospho-S6-S240, phospho-CAD, total CAD, and Tubulin expression in both paws and kidneys from wild type and Tsc1 cc DARPP Cre vehicle and rapamycin treated mice. D) Normalized quantification of expression of phospho-S6-240 relative to total S6-240 (top) and phospho-CAD relative to total CAD (bottom) in both paws and kidneys from wild type and Tsc1 cc DARPP Cre vehicle and rapamycin treated mice. n= 5 for phospho and total-S6-240 experimental groups, n=4 for each phospho and total-CAD experimental groups. * indicates P

    Article Snippet: Primary antibodies included P-CAD (Cell Signaling, Beverly, MA), CAD (Cell signaling), p-S6–240 (Cell Signaling), S6–240 (Cell Signaling), and anti-Tubulin (Abcam, Cambridge, UK).

    Techniques: Mouse Assay, Expressing

    Primary Keratinocytes Showed Impaired Proliferation upon  Glut1  Deletion (a) Glut1  mRNA (n=4 mice per genotype) and protein levels in the epidermis and primary keratinocytes harvested from one week old  WT, K14.Glut1 fl/wt ,  and  K14.Glut1  mice.  (b)  Immunostaining of Glut1 in skins from one-week old mice. Glut1 staining is absent in epidermis from  K14.Glut1  mice. Arrow indicates persistence of Glut1 staining in the dermis (e.g. dermal endothelial cell). Similar results obtained in 3 independent experiments.  (c)  mRNA abundance of  Glut/SGLT  family members in primary cultured  WT, Glut1 fl/wt K14Cre , and keratinocytes from  K14.Glut1  mice (n=4 mice per genotype).  (d)  2-Deoxy-D-glucose uptake in primary cultured keratinocytes obtained from one week old  WT, K14.Glut1 fl/wt ,  and  K14.Glut1  mice with or without glucose inhibitor pretreatment. Similar results obtained in uptake assays from keratinocytes from 3 independent mice.  (e)  Growth rate as assessed by Crystal Violet staining and relative cell number in primary  WT, K14.Glut1 fl/wt , and keratinocytes from  K14.Glut1  mice. Identical numbers of cells were seeded in triplicate wells in 6 well plates at day 0. For Crystal Violet staining, cells were stained 3 days after seeding and similar results were obtained in 3 keratinocytes preparations. Keratinocytes from  K14.Glut1  mice show significantly decreased growth compared to keratinoctyes from  WT  controls at day 1 and 2 after plating.  (f)  Glucose consumption and lactate production over 12 hours as measured from the media of primary cultured  WT  and  K14.Glut1  (n=3 mice per genoptye) keratinocytes.  (g)  ATP levels (luminescence) were determined in primary keratinocytes from  WT  and  K14.Glut1  (n=3 mice per genotype) and normalized to protein levels.  (h)  Western Blot analysis of the expression of p-ACC Ser79, ACC, p-S6 Ser240/244, Glut1 in primary keratinocytes obtained from  WT, Glut1 fl/wt K14Cre  and  K14.Glut1  mice. Data shown as mean±s.d.  P  values were calculated by two-tailed  t -test. Results were confirmed in at least 2 independent experiments.

    Journal: Nature medicine

    Article Title: Differential Glucose Requirement in Skin Homeostasis and Injury Identifies a Therapeutic Target for Psoriasis

    doi: 10.1038/s41591-018-0003-0

    Figure Lengend Snippet: Primary Keratinocytes Showed Impaired Proliferation upon Glut1 Deletion (a) Glut1 mRNA (n=4 mice per genotype) and protein levels in the epidermis and primary keratinocytes harvested from one week old WT, K14.Glut1 fl/wt , and K14.Glut1 mice. (b) Immunostaining of Glut1 in skins from one-week old mice. Glut1 staining is absent in epidermis from K14.Glut1 mice. Arrow indicates persistence of Glut1 staining in the dermis (e.g. dermal endothelial cell). Similar results obtained in 3 independent experiments. (c) mRNA abundance of Glut/SGLT family members in primary cultured WT, Glut1 fl/wt K14Cre , and keratinocytes from K14.Glut1 mice (n=4 mice per genotype). (d) 2-Deoxy-D-glucose uptake in primary cultured keratinocytes obtained from one week old WT, K14.Glut1 fl/wt , and K14.Glut1 mice with or without glucose inhibitor pretreatment. Similar results obtained in uptake assays from keratinocytes from 3 independent mice. (e) Growth rate as assessed by Crystal Violet staining and relative cell number in primary WT, K14.Glut1 fl/wt , and keratinocytes from K14.Glut1 mice. Identical numbers of cells were seeded in triplicate wells in 6 well plates at day 0. For Crystal Violet staining, cells were stained 3 days after seeding and similar results were obtained in 3 keratinocytes preparations. Keratinocytes from K14.Glut1 mice show significantly decreased growth compared to keratinoctyes from WT controls at day 1 and 2 after plating. (f) Glucose consumption and lactate production over 12 hours as measured from the media of primary cultured WT and K14.Glut1 (n=3 mice per genoptye) keratinocytes. (g) ATP levels (luminescence) were determined in primary keratinocytes from WT and K14.Glut1 (n=3 mice per genotype) and normalized to protein levels. (h) Western Blot analysis of the expression of p-ACC Ser79, ACC, p-S6 Ser240/244, Glut1 in primary keratinocytes obtained from WT, Glut1 fl/wt K14Cre and K14.Glut1 mice. Data shown as mean±s.d. P values were calculated by two-tailed t -test. Results were confirmed in at least 2 independent experiments.

    Article Snippet: For Western blotting, samples of cells or tissues were separated on SDS-PAGE gels, transferred to PVDF membranes and probed with primary antibodies as follows: Glut1 (1:2000, Thermo, RB-9052-P0), K10 (1:500, Santa Cruz, SC-70907), K14 (1:1000, Santa Cruz, SC-53253), HSP90 (1:1000, CST, 4877), Involucrin (1:1000, Thermo, MA5-11803), p-ACC Ser79 and ACC (1:1000, CST, 3661, 3662), p-S6 Ser240/244 (1:4000, CST, 5364), COX IV (1:2000, CST, 4844), and followed by secondary antibodies conjugated to HRP at a dilution of 1:5000 (Santa Cruz, Donkey anti-Rabbit: SC2077, Donkey anti-mouse: SC2096) and detection by the ECL System (PerkinElmer, NEL104001EA).

    Techniques: Mouse Assay, Immunostaining, Staining, Cell Culture, Western Blot, Expressing, Two Tailed Test

    Activation of cell-surface CD84 elevates PD-L1 levels through the p-AKT/mTOR pathway. ( A and B ) M210B4 cells (1 × 10 5 ) were stimulated with anti-CD84 (4 μg/ml) for 15 minutes, followed by anti-FAB cross-linking for 5 minutes. ( A ) Cells were lysed, and lysates were separated on 12% (wt/vol) SDS-PAGE and blotted with anti–p-S6, anti–p-AKT, anti–p-ERK, or actin. Blots are representative of 3 independent experiments. ( B ) Cells were fixed, permeabilized, and subsequently stained with anti–p-AKT and anti–p-S6 antibodies followed by a secondary anti-rabbit allophycocyanin-conjugated (APC-conjugated) antibody. Histograms are representative of 2 independent experiments. ( C – E ) Primary CLL cells were stimulated with control (IgG) or anti-CD84–activating (5 μg/ml) antibodies for 5 minutes, followed by anti-FAB cross-linking for an additional 5 minutes. Then, the cells were fixed, and p-ERK, p-AKT, and p-S6 levels were analyzed by flow cytometry ( n = 3; * P

    Journal: The Journal of Clinical Investigation

    Article Title: CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia

    doi: 10.1172/JCI96610

    Figure Lengend Snippet: Activation of cell-surface CD84 elevates PD-L1 levels through the p-AKT/mTOR pathway. ( A and B ) M210B4 cells (1 × 10 5 ) were stimulated with anti-CD84 (4 μg/ml) for 15 minutes, followed by anti-FAB cross-linking for 5 minutes. ( A ) Cells were lysed, and lysates were separated on 12% (wt/vol) SDS-PAGE and blotted with anti–p-S6, anti–p-AKT, anti–p-ERK, or actin. Blots are representative of 3 independent experiments. ( B ) Cells were fixed, permeabilized, and subsequently stained with anti–p-AKT and anti–p-S6 antibodies followed by a secondary anti-rabbit allophycocyanin-conjugated (APC-conjugated) antibody. Histograms are representative of 2 independent experiments. ( C – E ) Primary CLL cells were stimulated with control (IgG) or anti-CD84–activating (5 μg/ml) antibodies for 5 minutes, followed by anti-FAB cross-linking for an additional 5 minutes. Then, the cells were fixed, and p-ERK, p-AKT, and p-S6 levels were analyzed by flow cytometry ( n = 3; * P

    Article Snippet: The blots were probed with antibodies against actin (MilliporeSigma) as a housekeeping gene, anti–p-ERK, anti p-AKT, and anti–p-S6 (Cell Signaling Technology), and the bands were detected by Odyssey system (Li-Cor).

    Techniques: Activation Assay, SDS Page, Staining, Flow Cytometry, Cytometry

    Mean (± s.e.m. ) protein levels of p-mTOR, p-S6 kinase, ubiquitin and calpastatin in skeletal muscle from sheep fetuses on the fifth day of either saline ( n  = 6) or leptin ( n  = 6) infusion.

    Journal: The Journal of Physiology

    Article Title: Role of leptin in the regulation of growth and carbohydrate metabolism in the ovine fetus during late gestation

    doi: 10.1113/jphysiol.2007.149237

    Figure Lengend Snippet: Mean (± s.e.m. ) protein levels of p-mTOR, p-S6 kinase, ubiquitin and calpastatin in skeletal muscle from sheep fetuses on the fifth day of either saline ( n = 6) or leptin ( n = 6) infusion.

    Article Snippet: Polyclonal antibodies to IRβ, IGFIR, p110β and PKCζ were obtained from Santa Cruz Biotechnology (Santa Cruz, USA), to p85 from Upstate Biotechology (Millipore, Billerica, USA), to p-mTOR, p-S6 kinase and ubiquitin from Cell Signalling Technology (Danvers, USA), and to calpastatin from Abcam (Cambridge, UK).

    Techniques:

    In Vitro Response to Co-inhibition of MAPK and mTOR pathways in BRAF WT Glioma Cells (A–C) Cell viability responses to AZD6244 + everolimus assessed in (A) SF188 BRAF WT , ( B ) SF8628 BRAF WT and (C) SF9427 BRAF WT . For cell viability means of six replicates with standard errors are displayed. Measurements are normalized to DMSO control. Cell viability was assessed 72 hours after treatment. Pairwise comparisons were determined by Student’s t-test; * marks p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, and **** p-value ≤ 0.0001. ( D ) Apoptotic response of SF188 BRAF WT cells to combined inhibition of AZD6244 + everolimus. Apoptosis was measured by flow cytometry. The proportions of apoptotic cells were quantified 72 hours after treatment. All data points represent averages of at least four replicates. Statistical analysis includes ANOVA and Bonferroni multiple comparisons. ( E ) Cell cycle distribution of SF188 BRAF WT cells in response to combined inhibition of AZD6244 + everolimus. Analyses were performed 24 hours after treatment and depicted are averages of four replicates. Bar graphs display distribution of cells within each cell cycle phase. Error bars indicate standard error. (F) Western blot analyses of p-Akt, p-ERK and p-S6 in SF188 BRAF WT cells. β-actin was used as loading control. Levels of p-Akt, p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above results, in samples receiving combination therapy, inhibitors were administered simultaneously.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: BRAF status in personalizing treatment approaches for pediatric gliomas

    doi: 10.1158/1078-0432.CCR-15-1101

    Figure Lengend Snippet: In Vitro Response to Co-inhibition of MAPK and mTOR pathways in BRAF WT Glioma Cells (A–C) Cell viability responses to AZD6244 + everolimus assessed in (A) SF188 BRAF WT , ( B ) SF8628 BRAF WT and (C) SF9427 BRAF WT . For cell viability means of six replicates with standard errors are displayed. Measurements are normalized to DMSO control. Cell viability was assessed 72 hours after treatment. Pairwise comparisons were determined by Student’s t-test; * marks p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, and **** p-value ≤ 0.0001. ( D ) Apoptotic response of SF188 BRAF WT cells to combined inhibition of AZD6244 + everolimus. Apoptosis was measured by flow cytometry. The proportions of apoptotic cells were quantified 72 hours after treatment. All data points represent averages of at least four replicates. Statistical analysis includes ANOVA and Bonferroni multiple comparisons. ( E ) Cell cycle distribution of SF188 BRAF WT cells in response to combined inhibition of AZD6244 + everolimus. Analyses were performed 24 hours after treatment and depicted are averages of four replicates. Bar graphs display distribution of cells within each cell cycle phase. Error bars indicate standard error. (F) Western blot analyses of p-Akt, p-ERK and p-S6 in SF188 BRAF WT cells. β-actin was used as loading control. Levels of p-Akt, p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above results, in samples receiving combination therapy, inhibitors were administered simultaneously.

    Article Snippet: Five μm sections were cut, rehydrated with decreasing ethanol concentrations, and hybridized using anti-Ki67 (Dako, Carpinteria, CA), p-S6, p-Erk (R & D Systems, Minneapolis, MN), detected with DAB substrate (Vector Labs, CA) and counterstained with hematoxylin.

    Techniques: In Vitro, Inhibition, Flow Cytometry, Cytometry, Western Blot

    In Vitro Response to Co-inhibition of MAPK and mTOR pathways in BRAF V600E mutated DBTRG and AM-38 Glioma Cells Combination of (A) AZD6244 + everolimus, ( B) PLX4720 + everolimus and ( C) AZD6244 + PLX4720 in DBTRG cells. For each combination we display results of cell viability, apoptosis and cell cycle distribution (left to right). For cell viability, means of six replicates with standard errors are displayed. Measurements are normalized to DMSO control. Cell viability was assessed 72 hours after treatment. Pairwise comparisons were determined by Student’s t-test; * marks p-value ≤ 0.05; ** p-value ≤ 0.01, *** p-value ≤ 0.001, and **** p-value 0.0001. Apoptosis was measured by flow cytometry. The proportions of apoptotic cells were quantified 72 hours after treatment. All data points represent averages of at least four replicates. Statistical analysis include ANOVA and Bonferroni multiple comparisons. Cell cycle analyses were performed 24 hours after treatment and data points represent averages of four replicates. Bar graphs display distribution of cells within each cell cycle phase. Error bar indicate standard error. (D–F) Corresponding cell viability measurements in AM-38 for (D) AZD6244 + everolimus, (E) PLX4720 + everolimus and (F) AZD6244 + PLX4720. (G) Western blot analyses of p-Akt, p-ERK and p-S6 in BRAF V600E human glioma cells (DBTRG). β-Actin was used as loading control. Levels of p-Akt, p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above results, in samples receiving combination therapy, inhibitors were administered simultaneously.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: BRAF status in personalizing treatment approaches for pediatric gliomas

    doi: 10.1158/1078-0432.CCR-15-1101

    Figure Lengend Snippet: In Vitro Response to Co-inhibition of MAPK and mTOR pathways in BRAF V600E mutated DBTRG and AM-38 Glioma Cells Combination of (A) AZD6244 + everolimus, ( B) PLX4720 + everolimus and ( C) AZD6244 + PLX4720 in DBTRG cells. For each combination we display results of cell viability, apoptosis and cell cycle distribution (left to right). For cell viability, means of six replicates with standard errors are displayed. Measurements are normalized to DMSO control. Cell viability was assessed 72 hours after treatment. Pairwise comparisons were determined by Student’s t-test; * marks p-value ≤ 0.05; ** p-value ≤ 0.01, *** p-value ≤ 0.001, and **** p-value 0.0001. Apoptosis was measured by flow cytometry. The proportions of apoptotic cells were quantified 72 hours after treatment. All data points represent averages of at least four replicates. Statistical analysis include ANOVA and Bonferroni multiple comparisons. Cell cycle analyses were performed 24 hours after treatment and data points represent averages of four replicates. Bar graphs display distribution of cells within each cell cycle phase. Error bar indicate standard error. (D–F) Corresponding cell viability measurements in AM-38 for (D) AZD6244 + everolimus, (E) PLX4720 + everolimus and (F) AZD6244 + PLX4720. (G) Western blot analyses of p-Akt, p-ERK and p-S6 in BRAF V600E human glioma cells (DBTRG). β-Actin was used as loading control. Levels of p-Akt, p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above results, in samples receiving combination therapy, inhibitors were administered simultaneously.

    Article Snippet: Five μm sections were cut, rehydrated with decreasing ethanol concentrations, and hybridized using anti-Ki67 (Dako, Carpinteria, CA), p-S6, p-Erk (R & D Systems, Minneapolis, MN), detected with DAB substrate (Vector Labs, CA) and counterstained with hematoxylin.

    Techniques: In Vitro, Inhibition, Flow Cytometry, Cytometry, Western Blot

    In Vitro Response to Co-inhibition of MAPK and mTOR pathways in Isogenic Mouse BRAF V600E and BRAF WT Cells Assessments of isogenic murine (A–B) BRAF V600E and (C–D) BRAF WT cells in response to treatment with AZD6244 + everolimus, everolimus + PLX4720 or AZD6244 + PLX4720. ( A , C ) Represent cell viability data assessed 72 hours after treatment. Means of six replicates with standard errors are displayed. Measurements are normalized to DMSO controls. (B, D) Western blot analyses of p-Akt, p-ERK and p-S6 in isogenic murine (B) BRAF V600E and (D) BRAF WT cells. β-actin was used as loading control. Levels of p-Akt, p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above results, in samples receiving combination therapy, inhibitors were administered simultaneously.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: BRAF status in personalizing treatment approaches for pediatric gliomas

    doi: 10.1158/1078-0432.CCR-15-1101

    Figure Lengend Snippet: In Vitro Response to Co-inhibition of MAPK and mTOR pathways in Isogenic Mouse BRAF V600E and BRAF WT Cells Assessments of isogenic murine (A–B) BRAF V600E and (C–D) BRAF WT cells in response to treatment with AZD6244 + everolimus, everolimus + PLX4720 or AZD6244 + PLX4720. ( A , C ) Represent cell viability data assessed 72 hours after treatment. Means of six replicates with standard errors are displayed. Measurements are normalized to DMSO controls. (B, D) Western blot analyses of p-Akt, p-ERK and p-S6 in isogenic murine (B) BRAF V600E and (D) BRAF WT cells. β-actin was used as loading control. Levels of p-Akt, p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above results, in samples receiving combination therapy, inhibitors were administered simultaneously.

    Article Snippet: Five μm sections were cut, rehydrated with decreasing ethanol concentrations, and hybridized using anti-Ki67 (Dako, Carpinteria, CA), p-S6, p-Erk (R & D Systems, Minneapolis, MN), detected with DAB substrate (Vector Labs, CA) and counterstained with hematoxylin.

    Techniques: In Vitro, Inhibition, Western Blot

    In Vitro Response to Co-inhibition of MAPK and mTOR pathways in NIH/3T3 BRAF WT and KIAA1549:BRAF fusion cells (A, E) Cell viability in BRAF WT and KIAA1549:BRAF -expressing NIH/3T3 cells in response to AZD6244, everolimus or combination thereof under (A) high (10% DBS) and (E) low (0.5% DBS) serum conditions. Means of six replicates with standard error are displayed. Measurements are normalized to DMSO control. Cell viability was assessed 72 hours after treatment. (B, F) Cell cycle distribution of BRAF WT and KIAA1549:BRAF -expressing NIH/3T3 cells after treatment with AZD6244 and/or everolimus under (B) high (10%) and (F) low (0.5%) serum condition. Cell cycle analyses were performed 24 hours after treatment and depicted are averages of four experiments. Bar graphs display distributions of cells within each cell cycle phase. Error bars indicate standard error. Western blot analyses of p-Akt, p-ERK and p-S6 in (C, G) NIH/3T3 BRAF WT cells and (D, H) KIAA1549:BRAF -expressing NIH/3T3 cells after treatment with AZD6244, everolimus or combination thereof. β-actin was used as loading control. Levels of p-Akt, p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above results, in samples receiving combination therapy, inhibitors were administered simultaneously.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: BRAF status in personalizing treatment approaches for pediatric gliomas

    doi: 10.1158/1078-0432.CCR-15-1101

    Figure Lengend Snippet: In Vitro Response to Co-inhibition of MAPK and mTOR pathways in NIH/3T3 BRAF WT and KIAA1549:BRAF fusion cells (A, E) Cell viability in BRAF WT and KIAA1549:BRAF -expressing NIH/3T3 cells in response to AZD6244, everolimus or combination thereof under (A) high (10% DBS) and (E) low (0.5% DBS) serum conditions. Means of six replicates with standard error are displayed. Measurements are normalized to DMSO control. Cell viability was assessed 72 hours after treatment. (B, F) Cell cycle distribution of BRAF WT and KIAA1549:BRAF -expressing NIH/3T3 cells after treatment with AZD6244 and/or everolimus under (B) high (10%) and (F) low (0.5%) serum condition. Cell cycle analyses were performed 24 hours after treatment and depicted are averages of four experiments. Bar graphs display distributions of cells within each cell cycle phase. Error bars indicate standard error. Western blot analyses of p-Akt, p-ERK and p-S6 in (C, G) NIH/3T3 BRAF WT cells and (D, H) KIAA1549:BRAF -expressing NIH/3T3 cells after treatment with AZD6244, everolimus or combination thereof. β-actin was used as loading control. Levels of p-Akt, p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above results, in samples receiving combination therapy, inhibitors were administered simultaneously.

    Article Snippet: Five μm sections were cut, rehydrated with decreasing ethanol concentrations, and hybridized using anti-Ki67 (Dako, Carpinteria, CA), p-S6, p-Erk (R & D Systems, Minneapolis, MN), detected with DAB substrate (Vector Labs, CA) and counterstained with hematoxylin.

    Techniques: In Vitro, Inhibition, Expressing, Western Blot

    mTORC1 positively regulates OPN expression. (A) Tca8113 and KB cells were treated with or without 50 nM rapamycin (Rapa) for 24 h. Expression levels of OPN, p-S6 and S6 were detected by western blot analysis (top). The mRNA levels of OPN were analyzed by qRT-PCR (middle). OPN levels in the cell supernatants were detected by an ELISA (bottom). Data indicate mean ± SD of triplicate samples. ** P

    Journal: Journal of Cancer

    Article Title: Osteopontin is Critical for Hyperactive mTOR-Induced Tumorigenesis in Oral Squamous Cell Carcinoma

    doi: 10.7150/jca.18031

    Figure Lengend Snippet: mTORC1 positively regulates OPN expression. (A) Tca8113 and KB cells were treated with or without 50 nM rapamycin (Rapa) for 24 h. Expression levels of OPN, p-S6 and S6 were detected by western blot analysis (top). The mRNA levels of OPN were analyzed by qRT-PCR (middle). OPN levels in the cell supernatants were detected by an ELISA (bottom). Data indicate mean ± SD of triplicate samples. ** P

    Article Snippet: OPN and β-actin antibodies were purchased from Santa Cruz; p-S6 (Ser235/236), S6, mTOR, Raptor, and Rictor antibodies were obtained from Cell Signaling.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    RAPA inhibited the over-activation mTOR pathway to revers the senescence of MSCs from MRL/lpr mice ( A ) The over-expression of p-mTOR, p-S6K and p-S6 in p4 MSCs from MRL/lpr mice compared with normal group were determined by western blot analysis. The treatment of RAPA could obviously inhibit the expression of those proteins. GAPDH was used as the internal control. ( B ) P4 MSCs cultured into 24-well plates. The expression of p-mTOR, p-S6K and p-S6 analyzed by immunofluorescence staining showed that their over-activation in MSCs from MRL/lpr mice could be inhibited by RAPA treatment. Counterstaining with DAPI displays the localization of the nucleus (Scale bar = 50 μm). All data were expressed as the mean±SEM (n = 3,*P

    Journal: Aging (Albany NY)

    Article Title: Rapamycin reverses the senescent phenotype and improves immuno-regulation of mesenchymal stem cells from MRL/lpr mice and systemic lupus erythematosus patients through inhibition of the mTOR signaling pathway

    doi: 10.18632/aging.100925

    Figure Lengend Snippet: RAPA inhibited the over-activation mTOR pathway to revers the senescence of MSCs from MRL/lpr mice ( A ) The over-expression of p-mTOR, p-S6K and p-S6 in p4 MSCs from MRL/lpr mice compared with normal group were determined by western blot analysis. The treatment of RAPA could obviously inhibit the expression of those proteins. GAPDH was used as the internal control. ( B ) P4 MSCs cultured into 24-well plates. The expression of p-mTOR, p-S6K and p-S6 analyzed by immunofluorescence staining showed that their over-activation in MSCs from MRL/lpr mice could be inhibited by RAPA treatment. Counterstaining with DAPI displays the localization of the nucleus (Scale bar = 50 μm). All data were expressed as the mean±SEM (n = 3,*P

    Article Snippet: The following primary antibodies were used: GAPDH (anti-rabbit, Santa Cruz), p-mTOR (anti-rabbit, Sigma), mTOR (anti-rabbit, Sigma), p-S6K (anti-rabbit, Santa Cruz), S6K (anti-rabbit, Santa Cruz), p-S6 (anti-rabbit, Sigma), S6 (anti-rabbit, Sigma), p53 (anti-rabbit, Cell Signaling), p21 (anti-rabbit, Sigma), p27 (anti-rabbit, Cell Signaling).

    Techniques: Activation Assay, Mouse Assay, Over Expression, Western Blot, Expressing, Cell Culture, Immunofluorescence, Staining

    Over-activation of mTOR pathway was involved in the senescence of MSCs from SLE patients ( A ) The over-expression of p-mTOR, p-S6K and p-S6 in MSCs from SLE compared with normal group were determined by western blot analysis. GAPDH was used as the internal control. ( B ) P4 MSCs from SLE patients and normal group were cultured in 24-well plates. Immunofluorescence staining of p-mTOR, p-S6K and p-S6 verified their over-activation in SLE MSCs. Counterstaining with DAPI displays the localization of the nucleus (Scale bar = 50 μm). ( C - D ) P4 MSCs from SLE patients cultured at the different concentration of RAPA for 72 h. RAPA achieved maximal effects at about 500 nM by assaying the inhibition of p-70S6. (E-F) BMMSCs were depleted of mTOR by RNAi. The third one was chosen as the best siRNA by western blotting. ( G ) P4 MSCs from SLE patients were treated with 500 nM RAPA and si-mTOR or not for 72 h. Cellular morphology showed that the RAPA and si-mTOR treated SLE MSCs were less hypertrophic than untreated group (magnification; ×200). ( H ) MSCs were fixed and stained for β-gal. The number of SA-β-gal- positive cells obviously decreased among treated SLE MSCs in comparison with untreated group. ( I ) Immunofluorescence showed that RAPA and si-mTOR reversed abnormal F-actin distribution in MSCs from SLE patients. ( J ) P4 SLE MSCs were treated with 500 nM RAPA and the third si-mTOR or not, then transwell cultured with CD4+T cells for 72 h. The count of Treg cells decreased and Th17 cells increased in SLE MSCs compared to the normal group by flow cytometry analysis. But si-mTOR and RAPA-treated MSCs could reverse the abnormal changes. The statistical results revealed that RAPA could up-regulated the ratio of Treg/Th17 from SLE MSCs, which was down-regulated compared with normal group. ( K ) The supernatants of MSCs were collected. si-mTOR RAPA-treated SLE MSCs induced the secretion of IL-10 and TGF-β but reduced IL-17 and IL-6 by ELISA. All data were expressed as the mean±SEM (n = 3, *P

    Journal: Aging (Albany NY)

    Article Title: Rapamycin reverses the senescent phenotype and improves immuno-regulation of mesenchymal stem cells from MRL/lpr mice and systemic lupus erythematosus patients through inhibition of the mTOR signaling pathway

    doi: 10.18632/aging.100925

    Figure Lengend Snippet: Over-activation of mTOR pathway was involved in the senescence of MSCs from SLE patients ( A ) The over-expression of p-mTOR, p-S6K and p-S6 in MSCs from SLE compared with normal group were determined by western blot analysis. GAPDH was used as the internal control. ( B ) P4 MSCs from SLE patients and normal group were cultured in 24-well plates. Immunofluorescence staining of p-mTOR, p-S6K and p-S6 verified their over-activation in SLE MSCs. Counterstaining with DAPI displays the localization of the nucleus (Scale bar = 50 μm). ( C - D ) P4 MSCs from SLE patients cultured at the different concentration of RAPA for 72 h. RAPA achieved maximal effects at about 500 nM by assaying the inhibition of p-70S6. (E-F) BMMSCs were depleted of mTOR by RNAi. The third one was chosen as the best siRNA by western blotting. ( G ) P4 MSCs from SLE patients were treated with 500 nM RAPA and si-mTOR or not for 72 h. Cellular morphology showed that the RAPA and si-mTOR treated SLE MSCs were less hypertrophic than untreated group (magnification; ×200). ( H ) MSCs were fixed and stained for β-gal. The number of SA-β-gal- positive cells obviously decreased among treated SLE MSCs in comparison with untreated group. ( I ) Immunofluorescence showed that RAPA and si-mTOR reversed abnormal F-actin distribution in MSCs from SLE patients. ( J ) P4 SLE MSCs were treated with 500 nM RAPA and the third si-mTOR or not, then transwell cultured with CD4+T cells for 72 h. The count of Treg cells decreased and Th17 cells increased in SLE MSCs compared to the normal group by flow cytometry analysis. But si-mTOR and RAPA-treated MSCs could reverse the abnormal changes. The statistical results revealed that RAPA could up-regulated the ratio of Treg/Th17 from SLE MSCs, which was down-regulated compared with normal group. ( K ) The supernatants of MSCs were collected. si-mTOR RAPA-treated SLE MSCs induced the secretion of IL-10 and TGF-β but reduced IL-17 and IL-6 by ELISA. All data were expressed as the mean±SEM (n = 3, *P

    Article Snippet: The following primary antibodies were used: GAPDH (anti-rabbit, Santa Cruz), p-mTOR (anti-rabbit, Sigma), mTOR (anti-rabbit, Sigma), p-S6K (anti-rabbit, Santa Cruz), S6K (anti-rabbit, Santa Cruz), p-S6 (anti-rabbit, Sigma), S6 (anti-rabbit, Sigma), p53 (anti-rabbit, Cell Signaling), p21 (anti-rabbit, Sigma), p27 (anti-rabbit, Cell Signaling).

    Techniques: Activation Assay, Over Expression, Western Blot, Cell Culture, Immunofluorescence, Staining, Concentration Assay, Inhibition, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay