rapamycin Millipore Search Results


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  • 95
    Millipore rapamycin
    Differential regulation of autophagy by the two signalling pathways MEK/ERK and AKT/MTOR A. Western blot analysis of protein levels of pAKT (ser473), pS6R, pERKs, LC3 and tubulin after 24 h treatment with 1μM BRAFV600E inhibitor PLX4720 (lanes 2, 6 and 10), 1μM MEK inhibitor PD0325901 (lanes 3, 7 and 11) and 1μM of PI3K/MTOR inhibitor PI-103 (lanes 4, 8, 12), in the mutant BRAFV600E cell lines RKO, HT29 and Colo-205 respectively. The quantification of LC3 reflects the whole protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). B. The existence of autophagic vacuoles was analysed by 0,1 mM MDC staining under confocal microscope, after treatment of HT29 and Colo-205 cell lines with the kinase inhibitors, described in 3A. C. Cell viability of the mutant BRAFV600E colon cancer cell lines RKO, HT29 and Colo-205 after 24 (upper left panel), 48 (lower left panel) and 72 h (lower right panel) treatments with 1μM of each of the following BRAF/MEK/ERK and AKT/MTOR pathway inhibitors; PLX4720, PI-103, PD0325901 (PD), GDC0941 (GDC) (PI3K inhibitor), <t>Rapamycin</t> (MTOR inhibitor). Cells were also stained with Hoechst after 24h treatment in order to assess the number of apoptotic nuclei (upper right panel). D. Western blot analysis of LC3 and p62 protein levels in the mutant BRAFV600E colon cancer cell lines RKO, HT29 and Colo-205 respectively after 24 h treatment with 1μM of each of the following BRAF/MEK/ERK and AKT/MTOR pathway inhibitors: PLX4720 (2, 8, 14), PI-103 (3, 9, 15), PD0325901 (4, 10, 16), GDC0941 (5, 11, 17), Rapamycin (6, 12, 18). The quantification of LC3 reflects total protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). E. Confocal microscope images of two-dimensional culture in RKO cell line after treatment with 1μM of each of the following BRAF/MEK/ERK and PI3K/MTOR pathway inhibitors; PLX4720 (2 nd column), PI-103 (3 rd column), PD0325901(4 th column), GDC0941(5 th column), Rapamycin (6th column) as compared to control untreated cells (1st column). Cells were stained with MDC (upper row, light blue), phalloidin (middle row, red), and merged staining (lower row), in order to detect autophagic vacuoles (MDC) and cell number distribution. The number of cells stained with MDC (high-low) was recorded. The total number of cells by phalloidin staining and the number of MDC stained cells from five different confocal images for each sample was recorded. F. Western blot analysis of protein levels of pAKT(ser473), pS6R, LC3, p62 and tubulin after 24 h treatment with 1μM of PI-103 (lanes 2, 6, 10, 14), 1μM GDC0941 (lanes 3, 7, 11, 15) and 1μM Rapamycin (lanes 4, 8, 12, 16) in Caco-2, DLD-1, HCT116 and RKO cell lines. Protein levels were normalized against tubulin. The quantification of LC3 reflects the total protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). G. Confocal microscope images of two-dimensional culture in DLD-1 cell line after treatment with 1μM of each of the following PI3K/MTOR pathway inhibitors; PI-103 (2 nd column), GDC0941(3 rd column) and Rapamycin (4th column) as compared to control untreated cells (1st column). Cells were stained with Hoechst (upper row, blue), LC3 antibody (middle row, green) and merged (lower row). The total number of cells by Hoechst staining and the number of LC-3 stained cells from five different confocal images for each sample was recorded.
    Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 10231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc rapamycin
    <t>Rapamycin</t> treatment rescues the ciliary length phenotype of Tsc1 − / − cells. a IFM analysis of the expression of the proliferation marker KI67 and formation of primary cilia (arrows), labeled with anti-acetylated α-tubulin (Ac-TUB) antibody, in Tsc1 − / − MEFs cultured in normal serum medium (left panels), in starvation medium (0.5% FBS) for 48 h (middle panels, with few proliferating cells), or after 48 h starvation followed by serum re-stimulation for 24 h (right panels, with many proliferating cells). b WB analysis of WT MEFs subjected to siRNA-mediated knock down of Tsc1 or Tsc2 . WT MEFs were analyzed 48 h after transfection with siTsc1, siTsc2 or siScramble (negative control). GAPDH was used as loading control. c IFM analysis of primary cilia in WT MEFs after knock down of Tsc1 or Tsc2. Cilia (arrows) were labeled with Ac-TUB antibodies and the ciliary base/centrosomes (asterisks) were labeled with anti-γ-tubulin antibody (γ-TUB). Nuclei were visualized with DAPI staining. Scale bar: 5 µM. d Quantification of ciliary lengths for experiment shown in c . Three hundred cilia from three independent experiments were used for quantification; error bars represent SEM. e IFM analysis of primary cilia (Ac-TUB) in Tsc1 − / − and Tsc2 − / − MEFs subjected to siRNA-mediated knock down followed by culturing in starvation medium (0.5% serum) for 48 h (24 h after transfection) to induce cilia formation. Nuclei were visualized with DAPI staining. Scale bar: 5 µM. f Quantification of ciliary lengths for experiment shown in e . One hundred cilia from three independent experiments were used for quantification; error bars represent SEM. g Quantification of ciliary lengths labeled with Ac-TUB antibody in WT, Tsc1 − / − and Tsc2 − / − MEFs cultured in low 48 h in the presence or absence of rapamycin
    Rapamycin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1603 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore or rapamycin
    Effects of <t>rapamycin</t> or chloroquine on autophagic activity and EG-induced CaOx crystals depositions in rat kidneys. (A) Quantitative changes in autophagic vacuoles under TEM in kidney sections from indicated rats. The red arrows indicate autophagic vacuoles. Scale bar: 1 µm. (B) The expressions of Beclin1 and SQSTM1 in kidney sections from indicated rats were detected by immunohistochemical staining and quantified using Image Pro Plus software. (magnification 200×) (C) Photomicrographs of kidney sections from indicated rats were obtained under dark field illumination with polarized light. Retained crystals exhibit strong birefringence (magnification 200×). The sizes of the areas of crystal deposition per field were estimated and quantified using ImageJ software. * P
    Or Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Merck KGaA rapamycin
    LAM cell medium is chemotactic for fibroblasts, and this is partially dependent on CXCR4. (a) 621–101 cells secrete CXCL12 protein; this is partially sensitive to <t>rapamycin.</t> (b) The presence of 621–101 cell conditioned medium (CM) increases fibroblast migration, which is partially blocked by AMD3100. (c) Fibroblasts migrate towards CXCL12 in a modified Boyden chamber (Transwell) assay; this is inhibited by the CXCR4 receptor antagonist AMD3100 at 100μg/ml (*p = 0.05). (d) Fibroblasts migrate towards 621–101 cells in a bidirectional migration assay. (i) Removal of the fence leaves a gap between the peripheral fibroblasts (green) and the central reservoir of (red) 621–101 cells (dotted), (ii) Fibroblasts migrate towards the central reservoir (arrowed), (iii) fibroblasts migrate into the central area (arrowed). (e) Addition of 200μg/ml AMD3100 reduces migration of fibroblasts in response to 621–101 cells by 56%. In the absence of 621–101 cells in the central reservoir migration is reduced by 93%.
    Rapamycin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 97/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore completerpmi1640 rapamycin
    LAM cell medium is chemotactic for fibroblasts, and this is partially dependent on CXCR4. (a) 621–101 cells secrete CXCL12 protein; this is partially sensitive to <t>rapamycin.</t> (b) The presence of 621–101 cell conditioned medium (CM) increases fibroblast migration, which is partially blocked by AMD3100. (c) Fibroblasts migrate towards CXCL12 in a modified Boyden chamber (Transwell) assay; this is inhibited by the CXCR4 receptor antagonist AMD3100 at 100μg/ml (*p = 0.05). (d) Fibroblasts migrate towards 621–101 cells in a bidirectional migration assay. (i) Removal of the fence leaves a gap between the peripheral fibroblasts (green) and the central reservoir of (red) 621–101 cells (dotted), (ii) Fibroblasts migrate towards the central reservoir (arrowed), (iii) fibroblasts migrate into the central area (arrowed). (e) Addition of 200μg/ml AMD3100 reduces migration of fibroblasts in response to 621–101 cells by 56%. In the absence of 621–101 cells in the central reservoir migration is reduced by 93%.
    Completerpmi1640 Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rapamycin mtor rapamycin
    Targeting FAK via RNAi enhanced the growth-inhibitory effects of <t>rapamycin.</t> a FAK protein ( i ) and mRNA expression ( ii ) in REH-empty vector and REH-FAK shRNA cells. b The <t>mTOR</t> inhibitor rapamycin and FAK down-regulation inhibited the proliferation of REH cells. REH-empty vector or REH-FAK shRNA cells were plated in 96-well plates and cultured with rapamycin (0–1000 nM). After 2 days ( i ) or 3 days ( ii ), the cells were treated with CCK-8 (10 μl/well) for 2 h and the absorbance was measured. The results represent the mean ± S.D. of three experiments performed in triplicate
    Rapamycin Mtor Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore ηm rapamycin
    Growth and cell cycle regulation during endothelial differentiation and <t>rapamycin</t> treatment . a) Rate of cell division. RoSH2 cells were labeled with CFDA, a cell-permeant fluorescent dye, cultured for 24 hours and re-plated on gelatin-coated plates to be maintained as undifferentiated cells (Undif) or on matrigel to induce differentiation (Dif). Cells were harvested at 0, 24, 48 and 72 hours. At 48 hours after replating, half of the remaining plates of cells under undifferentiating condition or differentiating condition were treated with 50 ηM rapamycin (R undif and Rdif, respectively). Median cellular fluorescence of the harvested cells was measured by flow cytometry and the number of cell divisions was calculated as a function of the loss in fluorescence; b) Cell cycle progression during endothelial differentiation. RoSH2 cells were plated on either gelatin-coated plate (self-renewing) or matrigel (differentiating) and labeled with BrdU for 16 hours. After removing BrdU, half of the gelatin-coated plates were treated with 50 ηM rapamycin. At 0, 6 and 12 hours, cells were harvested, stained with anti-BrdU and PI. DNA content of BrdU-labeled cells as measured by PI was analyzed by flow cytometry.
    ηm Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore rapamycin mtor inhibitor rapamycin
    Immunoblot analysis of (A) Hela, (B) Caki-1 and (C) Hepa-1 cells. Cells were exposed to either normoxia (N, 21% O 2 ), hypoxia (H, 1% O 2 ) or DP (Dipyridyl, 100µM) treatment for 6 or 24 hours. 30µg of total cell lysate was loaded and western blotting was performed for the following proteins: HIF-1α, HIF-2α, p70S6K-P (Thr 389), p70S6K, rpS6-P (Ser 235/236), rpS6 and ß-actin. Hepa-1 cells are either wildtype cells (Hepa-1 C1C7) or HIF-1ß deficient (Hepa-1 C4). Therefore, these cells were additionally analyzed for the expression of HIF-1ß. The <t>mTOR</t> inhibitor <t>rapamycin</t> was applied 45 minutes prior to hypoxic/pharmacological exposure at a concentration of <t>100nM.</t>
    Rapamycin Mtor Inhibitor Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore s6k inhibitor rapamycin
    Immunoblot analysis of (A) Hela, (B) Caki-1 and (C) Hepa-1 cells. Cells were exposed to either normoxia (N, 21% O 2 ), hypoxia (H, 1% O 2 ) or DP (Dipyridyl, 100µM) treatment for 6 or 24 hours. 30µg of total cell lysate was loaded and western blotting was performed for the following proteins: HIF-1α, HIF-2α, p70S6K-P (Thr 389), p70S6K, rpS6-P (Ser 235/236), rpS6 and ß-actin. Hepa-1 cells are either wildtype cells (Hepa-1 C1C7) or HIF-1ß deficient (Hepa-1 C4). Therefore, these cells were additionally analyzed for the expression of HIF-1ß. The <t>mTOR</t> inhibitor <t>rapamycin</t> was applied 45 minutes prior to hypoxic/pharmacological exposure at a concentration of <t>100nM.</t>
    S6k Inhibitor Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    LC Laboratories rapamycin
    Immunoblot analysis of (A) Hela, (B) Caki-1 and (C) Hepa-1 cells. Cells were exposed to either normoxia (N, 21% O 2 ), hypoxia (H, 1% O 2 ) or DP (Dipyridyl, 100µM) treatment for 6 or 24 hours. 30µg of total cell lysate was loaded and western blotting was performed for the following proteins: HIF-1α, HIF-2α, p70S6K-P (Thr 389), p70S6K, rpS6-P (Ser 235/236), rpS6 and ß-actin. Hepa-1 cells are either wildtype cells (Hepa-1 C1C7) or HIF-1ß deficient (Hepa-1 C4). Therefore, these cells were additionally analyzed for the expression of HIF-1ß. The <t>mTOR</t> inhibitor <t>rapamycin</t> was applied 45 minutes prior to hypoxic/pharmacological exposure at a concentration of <t>100nM.</t>
    Rapamycin, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 3393 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tor inhibitor rapamycin
    <t>TOR</t> signaling is required for early trypsin protein synthesis in amino acid fed mosquitoes. A) Injection of TOR dsRNA had no effect on early trypsin transcript levels in unfed mosquitoes based on quantitative RT-PCR. B) Injection of TOR dsRNA or <t>rapamycin</t>
    Tor Inhibitor Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore torc1 inhibitor rapamycin
    IGF-I dependent DDR1 protein upregulation is downstream the PI3K/AKT pathway and does not require activation of the ERK1/2 and the mTOR pathways MCF-7 cells grown with serum stripped 2.5% of FCS for 24 h, were pretreated with various kinase inhibitors at the indicated doses for 1 h. Cells were then stimulated with 50 nM IGF-I for 24 h, lysed and analyzed by western blotting to evaluate DDR1 protein expression. ( A ) Cells treated with the PI3K inhibitor LY294002 (LY), and ( B ) the AKT inhibitor1/2 (iAKT). ( C ) Cells treated with the MEK1 inhibitor U0126, and ( D ) with the <t>TORC1</t> inhibitor <t>Rapamycin</t> (Rapa). Immunoblot for α-tubulin was used as control for protein loading. Each blot shown is representative of three independent experiments. Values are mean ± SEM of three separate experiments. * P
    Torc1 Inhibitor Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore autophagy inducer rapamycin
    Induction of drug resistance in HMEC-1 cells and sunitinib-induced activation of autophagy. (A) Replication times of cultured HMEC-1 cells in the presence of indicated concentrations of sunitinib. Data were calculated from more than two repetitions. (B) Evolution of the IC50 of sunitinib in HMEC-1 cells after treatment with 6 µ M sunitinib for different time periods. (C) Plotted survival rates of HMEC-1 cells and sunitinib-resistant HMEC 6 µ M cells. Normal HMEC-1 cells and sunitinib-resistant HMEC 6 µ M cells were exposed to the indicated concentrations of sunitinib for 72 h, and the cells were trypsinized and counted. Non-treated cells were set as 100%. (A-C) Curves are derived from the means of more than three experiments. (D) LysoTracker DND-99 density of HMEC-1 cells following incubation with 6 µ M sunitinib for different time periods. Cells were treated with sunitinib for 24 or 48 h and LysoTracker DND-99 was added followed by flow cytometric analysis. (E) LysoTracker DND-99 density of sunitinib-treated HMEC-1 cells and HMEC 6 µ M cells. The cells were washed five times and then incubated with LysoTracker DND-99. Fluorescence was measured with the control cells without LysoTracker DND-99 staining using flow cytometry. (F-H) Western blot analysis using anti-LAMP-1, anti-LC3 and anti-p62 antibodies. HMEC-1 and HMEC 6 µ M cells were exposed to 6 µ M sunitinib, or autophagy inducer 5 µ M <t>rapamycin,</t> or Hanks' media for 24 h. Subsequently, the cells were collected and extracted proteins were used for western blotting. The experiments were repeated at least three times. * P
    Autophagy Inducer Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore autophagy activator rapamycin
    Immunohistochemical analysis of the effects of LV-Atg7 treatment in α-syn tg mice. For panels A–M, vibratome sections from non tg and α-syn tg mice that received LV injections into the cortex and hippocampus were immunolabeled with antibodies against Atg7 or α-syn and imaged with a digital microscope. Panels A′–M′ represent higher-power images from the hippocampus of the corresponding low-power panels in panels A–M. For panels O–T, effects of <t>rapamycin</t> treatment on α-syn accumulation, autophagy and neuronal integrity in the brains of α-syn tg mice. For panels A–M, vibratome sections from the hippocampus of non tg and α-syn tg mice were immunolabeled with an antibody against MAP2 and imaged with a laser scanning confocal microscope, and images were obtained from the temporal cortex. (A–F) Representative sections from the brains of non tg (A, B) and α-syn tg mice (C–F) that received injections with LV-control (A–D) or LV-Atg7 (E, F) and were immunolabeled with an antibody against Atg7. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (G) Semi-quantitative image analysis of Atg7 immunoreactivity in non tg and α-syn tg mice show increased Atg7 levels ipsilateral to the injection sites in the brains of animals that received LV-Atg7. (H–M) Representative sections from the brains of non tg (H, I) and α-syn tg mice (J–M) that received injections with LV-control (H–K) or LV-Atg7 (L, M) and were immunolabeled with an antibody against α-syn. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (N) Semi-quantitative image analysis of α-syn immunoreactivity in non tg and α-syn tg mice show reduced α-syn levels ipsilateral to the injection sites in the brains of α-syn tg mice that received LV-Atg7 injections. (O–T) Representative sections from the brains of non tg (O, P) and α-syn tg mice (Q–T) that received injections with LV-control (O–R) or LV-Atg7 (S, T) and were immunolabeled with an antibody against MAP2. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (U) Semi-quantitative image analysis of MAP2 immunoreactivity in non tg and α-syn tg mice shows a recovery of MAP2 levels ipsilateral to the injection sites in the brains of α-syn tg mice that received LV-Atg7 injections. Scale bar in panel (F) represents 0.1mm in panels A–F and H–M, 20µm in panels A′–F′ and H′–M′, and 10µm in panels O–T. *p
    Autophagy Activator Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rapamycin complex 1 mtorc1 inhibitor rapamycin
    Immunohistochemical analysis of the effects of LV-Atg7 treatment in α-syn tg mice. For panels A–M, vibratome sections from non tg and α-syn tg mice that received LV injections into the cortex and hippocampus were immunolabeled with antibodies against Atg7 or α-syn and imaged with a digital microscope. Panels A′–M′ represent higher-power images from the hippocampus of the corresponding low-power panels in panels A–M. For panels O–T, effects of <t>rapamycin</t> treatment on α-syn accumulation, autophagy and neuronal integrity in the brains of α-syn tg mice. For panels A–M, vibratome sections from the hippocampus of non tg and α-syn tg mice were immunolabeled with an antibody against MAP2 and imaged with a laser scanning confocal microscope, and images were obtained from the temporal cortex. (A–F) Representative sections from the brains of non tg (A, B) and α-syn tg mice (C–F) that received injections with LV-control (A–D) or LV-Atg7 (E, F) and were immunolabeled with an antibody against Atg7. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (G) Semi-quantitative image analysis of Atg7 immunoreactivity in non tg and α-syn tg mice show increased Atg7 levels ipsilateral to the injection sites in the brains of animals that received LV-Atg7. (H–M) Representative sections from the brains of non tg (H, I) and α-syn tg mice (J–M) that received injections with LV-control (H–K) or LV-Atg7 (L, M) and were immunolabeled with an antibody against α-syn. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (N) Semi-quantitative image analysis of α-syn immunoreactivity in non tg and α-syn tg mice show reduced α-syn levels ipsilateral to the injection sites in the brains of α-syn tg mice that received LV-Atg7 injections. (O–T) Representative sections from the brains of non tg (O, P) and α-syn tg mice (Q–T) that received injections with LV-control (O–R) or LV-Atg7 (S, T) and were immunolabeled with an antibody against MAP2. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (U) Semi-quantitative image analysis of MAP2 immunoreactivity in non tg and α-syn tg mice shows a recovery of MAP2 levels ipsilateral to the injection sites in the brains of α-syn tg mice that received LV-Atg7 injections. Scale bar in panel (F) represents 0.1mm in panels A–F and H–M, 20µm in panels A′–F′ and H′–M′, and 10µm in panels O–T. *p
    Rapamycin Complex 1 Mtorc1 Inhibitor Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA mtorc1 inhibitor rapamycin
    Raptor siRNA mimics the effects of rapa, and mammalian target of <t>rapamycin</t> complex 1 inhibition induces HIF2A expression. (A): SOX9 protein expression levels of chondrogenic pellets cultured in differentiation medium (control) containing 25 nM rapa or
    Mtorc1 Inhibitor Rapamycin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rapamycin mtor inhibitor inhibitor rapamycin 100 nm
    Raptor siRNA mimics the effects of rapa, and mammalian target of <t>rapamycin</t> complex 1 inhibition induces HIF2A expression. (A): SOX9 protein expression levels of chondrogenic pellets cultured in differentiation medium (control) containing 25 nM rapa or
    Rapamycin Mtor Inhibitor Inhibitor Rapamycin 100 Nm, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore macroautophagy activator rapamycin
    Raptor siRNA mimics the effects of rapa, and mammalian target of <t>rapamycin</t> complex 1 inhibition induces HIF2A expression. (A): SOX9 protein expression levels of chondrogenic pellets cultured in differentiation medium (control) containing 25 nM rapa or
    Macroautophagy Activator Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SH-SY5Y cells morphological changes during osteogenic differentiation. SH-SY5Y cells were cultured in osteogenic medium in the presence of hydroxyapatite scaffolds with <t>rapamycin</t> (5 μ M) and analyzed for osteocalcin expression by immunocytochemistry. ( a ) SH-SY5Y cells on the scaffolds during differentiation (in green, osteocalcin; in blue, DAPI; bar=50 μ m). ( b ) Osteocalcin secretion by SH-SY5Y during differentiation (in green, osteocalcin; in blue, DAPI; bar=50 μ m)
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    Effects of autophagy regulation and oxygen-glucose deprivation <t>(OGD)</t> on BACE1 expression in Neuro-2a/amyloid precursor protein 695 cells (western blot assay). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with <t>Rapamycin</t> (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-methyladenine (5 mM). * P
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    In vivo chemical dimerization facilitates microtubule plus end tracking in real time HeLa cells dually transfected with FRB-EGFP and FKBP-EGFP fusions of EB1 and/or CLIP-170. EB1 constructs embody residues 1-185, CLIP-170 constructs residues 3-210 and 129-350. After one minute of imaging, <t>rapamycin</t> was added to the media for a final concentration of 50 nM. Magnified images at right correspond to the boxed region in the first column of the respective row. Images represent a single time point pre-rapamycin treatment and three consecutive time points, taken at 2 sec intervals, post-rapamycin treatment. Arrows, color-coded to the respective time point, track individual microtubule plus ends. Time in sec is indicated relative to rapamycin addition (t = 0 sec). Scale bar in left column, 5 μm.
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    <t>Rapamycin</t> rescues laminin α 2 knockdown-mediated changes in the spatial expression of actin binding and regulatory proteins. Sertoli cells cultured for 3 days were transfected with laminin α 2 (Lam α 2) vs negative control (Ctrl) shRNA for 24 hours. Cells were rinsed and treated with rapamycin (100 ng/mL) for 24 hours (for IF). For IB, transfected cells were cultured for an additional 24 hours and were then treated with rapamycin (100 ng/mL) for 24 hours. (a) A study by IB showed that laminin α 2 knockdown and/or rapamycin had no apparent effect on the Arp3 and Eps8 steady-state protein levels. β -actin served as a protein-loading control. (b) A study by IF illustrated that laminin α 2 knockdown caused redistribution of branched actin polymerization protein Arp3 and actin barbed end capping and bundling protein Eps8. These two proteins no longer prominently localized at the cell-cell interface to maintain proper organization of actin filament bundles to support the Sertoli cell TJ barrier function following laminin α 2 knockdown. These proteins were mostly internalized after laminin α 2 knockdown. However, rapamycin blocked laminin α 2 knockdown-induced redistribution of Arp3 and Eps8 in Sertoli cells. GFP expression (green) illustrated successful transfection. Sertoli cell nuclei were visualized by DAPI. Scale bar, 30 μm.
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    Bace2 regulates melanophore dendricity via PI3K/mTOR signaling (A) Scheme for chemical suppressor screen of the bace2 −/− mutant. 24hpf bace2 −/− embryos were treated with each compound from the Sigma LOPAC 1280 library at 30μM for 48 hours, in order to identify chemicals which could rescue the melanophore defects. (B) Compounds were scored with a range of 0 (non-rescued, mutant-like) to 5 (fully rescued, WT-like). (C) Top hits from the screen (with score of 4 and 5) converge on PI3K/mTOR signaling pathway. PI3K inhibitors AS605240 (110nM), Wortmannin (230nM), LY-294,002 (15μM), and mTOR inhibitors <t>Temsirolimus</t> (30μM), PP242 (15μM) all fully rescue the bace2 −/− hyperdendritic melanophores. (D) Quantification of tailfin melanophore cell area at 72hpf with hits from the screen (n=each fish, one-way ANOVA followed by Holm-Sidak’s multiple comparisons test, ****P
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    Bace2 regulates melanophore dendricity via PI3K/mTOR signaling (A) Scheme for chemical suppressor screen of the bace2 −/− mutant. 24hpf bace2 −/− embryos were treated with each compound from the Sigma LOPAC 1280 library at 30μM for 48 hours, in order to identify chemicals which could rescue the melanophore defects. (B) Compounds were scored with a range of 0 (non-rescued, mutant-like) to 5 (fully rescued, WT-like). (C) Top hits from the screen (with score of 4 and 5) converge on PI3K/mTOR signaling pathway. PI3K inhibitors AS605240 (110nM), Wortmannin (230nM), LY-294,002 (15μM), and mTOR inhibitors <t>Temsirolimus</t> (30μM), PP242 (15μM) all fully rescue the bace2 −/− hyperdendritic melanophores. (D) Quantification of tailfin melanophore cell area at 72hpf with hits from the screen (n=each fish, one-way ANOVA followed by Holm-Sidak’s multiple comparisons test, ****P
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    Enhanced Akt-rpS6 activation and in vitro inhibition of rpS6 activation in Oo Pten −/− oocytes by <t>rapamycin.</t> ( A ) Comparison of Akt-rpS6 signaling in Oo Pten −/− and Oo Pten +/+ oocytes. Oocytes were isolated from ovaries of mice at postnatal day 12–14 and immunoblotting was performed as described in Materials and Methods . Loss of PTEN led to enhanced PI3K signaling as indicated by an increase in phosphorylated Akt (p-Akt). The level of phosphorylated rpS6 (p-rpS6) was also increased in Oo Pten −/− oocytes compared with Oo Pten +/+ oocytes. Levels of total rpS6, Akt, and β-actin were used as internal controls. ( B ) Activation of rpS6 in Oo Pten −/− oocytes is dependent on <t>mTORC1</t> signaling. Oocytes were isolated from ovaries of Oo Pten −/− mice at PD 12–14 as described in Materials and Methods . Treatment of oocytes with the mTORC1-specific inhibitor rapamycin (Rapa, 50 nM) for 2 h was found to largely suppress levels of phosphorylated rpS6 (p-rpS6), but did not affect the level of phosphorylated Akt (p-Akt). As a control, treatment of Oo Pten −/− oocytes with the PI3K-specific inhibitor LY294002 (LY, 50 µM) for 2 h also largely suppressed levels of phosphorylated rpS6 (p-rpS6), but it also suppressed the level of phosphorylated Akt (p-Akt). This suggests that activation of rpS6 in Oo Pten −/− oocytes is dependent on both PI3K and mTORC1 signaling. Levels of total Akt, rpS6, and β-actin were used as internal controls.
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    Enhanced Akt-rpS6 activation and in vitro inhibition of rpS6 activation in Oo Pten −/− oocytes by <t>rapamycin.</t> ( A ) Comparison of Akt-rpS6 signaling in Oo Pten −/− and Oo Pten +/+ oocytes. Oocytes were isolated from ovaries of mice at postnatal day 12–14 and immunoblotting was performed as described in Materials and Methods . Loss of PTEN led to enhanced PI3K signaling as indicated by an increase in phosphorylated Akt (p-Akt). The level of phosphorylated rpS6 (p-rpS6) was also increased in Oo Pten −/− oocytes compared with Oo Pten +/+ oocytes. Levels of total rpS6, Akt, and β-actin were used as internal controls. ( B ) Activation of rpS6 in Oo Pten −/− oocytes is dependent on <t>mTORC1</t> signaling. Oocytes were isolated from ovaries of Oo Pten −/− mice at PD 12–14 as described in Materials and Methods . Treatment of oocytes with the mTORC1-specific inhibitor rapamycin (Rapa, 50 nM) for 2 h was found to largely suppress levels of phosphorylated rpS6 (p-rpS6), but did not affect the level of phosphorylated Akt (p-Akt). As a control, treatment of Oo Pten −/− oocytes with the PI3K-specific inhibitor LY294002 (LY, 50 µM) for 2 h also largely suppressed levels of phosphorylated rpS6 (p-rpS6), but it also suppressed the level of phosphorylated Akt (p-Akt). This suggests that activation of rpS6 in Oo Pten −/− oocytes is dependent on both PI3K and mTORC1 signaling. Levels of total Akt, rpS6, and β-actin were used as internal controls.
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    Image Search Results


    Differential regulation of autophagy by the two signalling pathways MEK/ERK and AKT/MTOR A. Western blot analysis of protein levels of pAKT (ser473), pS6R, pERKs, LC3 and tubulin after 24 h treatment with 1μM BRAFV600E inhibitor PLX4720 (lanes 2, 6 and 10), 1μM MEK inhibitor PD0325901 (lanes 3, 7 and 11) and 1μM of PI3K/MTOR inhibitor PI-103 (lanes 4, 8, 12), in the mutant BRAFV600E cell lines RKO, HT29 and Colo-205 respectively. The quantification of LC3 reflects the whole protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). B. The existence of autophagic vacuoles was analysed by 0,1 mM MDC staining under confocal microscope, after treatment of HT29 and Colo-205 cell lines with the kinase inhibitors, described in 3A. C. Cell viability of the mutant BRAFV600E colon cancer cell lines RKO, HT29 and Colo-205 after 24 (upper left panel), 48 (lower left panel) and 72 h (lower right panel) treatments with 1μM of each of the following BRAF/MEK/ERK and AKT/MTOR pathway inhibitors; PLX4720, PI-103, PD0325901 (PD), GDC0941 (GDC) (PI3K inhibitor), Rapamycin (MTOR inhibitor). Cells were also stained with Hoechst after 24h treatment in order to assess the number of apoptotic nuclei (upper right panel). D. Western blot analysis of LC3 and p62 protein levels in the mutant BRAFV600E colon cancer cell lines RKO, HT29 and Colo-205 respectively after 24 h treatment with 1μM of each of the following BRAF/MEK/ERK and AKT/MTOR pathway inhibitors: PLX4720 (2, 8, 14), PI-103 (3, 9, 15), PD0325901 (4, 10, 16), GDC0941 (5, 11, 17), Rapamycin (6, 12, 18). The quantification of LC3 reflects total protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). E. Confocal microscope images of two-dimensional culture in RKO cell line after treatment with 1μM of each of the following BRAF/MEK/ERK and PI3K/MTOR pathway inhibitors; PLX4720 (2 nd column), PI-103 (3 rd column), PD0325901(4 th column), GDC0941(5 th column), Rapamycin (6th column) as compared to control untreated cells (1st column). Cells were stained with MDC (upper row, light blue), phalloidin (middle row, red), and merged staining (lower row), in order to detect autophagic vacuoles (MDC) and cell number distribution. The number of cells stained with MDC (high-low) was recorded. The total number of cells by phalloidin staining and the number of MDC stained cells from five different confocal images for each sample was recorded. F. Western blot analysis of protein levels of pAKT(ser473), pS6R, LC3, p62 and tubulin after 24 h treatment with 1μM of PI-103 (lanes 2, 6, 10, 14), 1μM GDC0941 (lanes 3, 7, 11, 15) and 1μM Rapamycin (lanes 4, 8, 12, 16) in Caco-2, DLD-1, HCT116 and RKO cell lines. Protein levels were normalized against tubulin. The quantification of LC3 reflects the total protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). G. Confocal microscope images of two-dimensional culture in DLD-1 cell line after treatment with 1μM of each of the following PI3K/MTOR pathway inhibitors; PI-103 (2 nd column), GDC0941(3 rd column) and Rapamycin (4th column) as compared to control untreated cells (1st column). Cells were stained with Hoechst (upper row, blue), LC3 antibody (middle row, green) and merged (lower row). The total number of cells by Hoechst staining and the number of LC-3 stained cells from five different confocal images for each sample was recorded.

    Journal: Oncotarget

    Article Title: BRAF associated autophagy exploitation: BRAF and autophagy inhibitors synergise to efficiently overcome resistance of BRAF mutant colorectal cancer cells

    doi: 10.18632/oncotarget.6942

    Figure Lengend Snippet: Differential regulation of autophagy by the two signalling pathways MEK/ERK and AKT/MTOR A. Western blot analysis of protein levels of pAKT (ser473), pS6R, pERKs, LC3 and tubulin after 24 h treatment with 1μM BRAFV600E inhibitor PLX4720 (lanes 2, 6 and 10), 1μM MEK inhibitor PD0325901 (lanes 3, 7 and 11) and 1μM of PI3K/MTOR inhibitor PI-103 (lanes 4, 8, 12), in the mutant BRAFV600E cell lines RKO, HT29 and Colo-205 respectively. The quantification of LC3 reflects the whole protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). B. The existence of autophagic vacuoles was analysed by 0,1 mM MDC staining under confocal microscope, after treatment of HT29 and Colo-205 cell lines with the kinase inhibitors, described in 3A. C. Cell viability of the mutant BRAFV600E colon cancer cell lines RKO, HT29 and Colo-205 after 24 (upper left panel), 48 (lower left panel) and 72 h (lower right panel) treatments with 1μM of each of the following BRAF/MEK/ERK and AKT/MTOR pathway inhibitors; PLX4720, PI-103, PD0325901 (PD), GDC0941 (GDC) (PI3K inhibitor), Rapamycin (MTOR inhibitor). Cells were also stained with Hoechst after 24h treatment in order to assess the number of apoptotic nuclei (upper right panel). D. Western blot analysis of LC3 and p62 protein levels in the mutant BRAFV600E colon cancer cell lines RKO, HT29 and Colo-205 respectively after 24 h treatment with 1μM of each of the following BRAF/MEK/ERK and AKT/MTOR pathway inhibitors: PLX4720 (2, 8, 14), PI-103 (3, 9, 15), PD0325901 (4, 10, 16), GDC0941 (5, 11, 17), Rapamycin (6, 12, 18). The quantification of LC3 reflects total protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). E. Confocal microscope images of two-dimensional culture in RKO cell line after treatment with 1μM of each of the following BRAF/MEK/ERK and PI3K/MTOR pathway inhibitors; PLX4720 (2 nd column), PI-103 (3 rd column), PD0325901(4 th column), GDC0941(5 th column), Rapamycin (6th column) as compared to control untreated cells (1st column). Cells were stained with MDC (upper row, light blue), phalloidin (middle row, red), and merged staining (lower row), in order to detect autophagic vacuoles (MDC) and cell number distribution. The number of cells stained with MDC (high-low) was recorded. The total number of cells by phalloidin staining and the number of MDC stained cells from five different confocal images for each sample was recorded. F. Western blot analysis of protein levels of pAKT(ser473), pS6R, LC3, p62 and tubulin after 24 h treatment with 1μM of PI-103 (lanes 2, 6, 10, 14), 1μM GDC0941 (lanes 3, 7, 11, 15) and 1μM Rapamycin (lanes 4, 8, 12, 16) in Caco-2, DLD-1, HCT116 and RKO cell lines. Protein levels were normalized against tubulin. The quantification of LC3 reflects the total protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). G. Confocal microscope images of two-dimensional culture in DLD-1 cell line after treatment with 1μM of each of the following PI3K/MTOR pathway inhibitors; PI-103 (2 nd column), GDC0941(3 rd column) and Rapamycin (4th column) as compared to control untreated cells (1st column). Cells were stained with Hoechst (upper row, blue), LC3 antibody (middle row, green) and merged (lower row). The total number of cells by Hoechst staining and the number of LC-3 stained cells from five different confocal images for each sample was recorded.

    Article Snippet: PI-103 #S1038 SELLECKCHEM, Rapamycin #R8781 SIGMA, GDC0941 #S1065 SELLECKCHEM, PD 0325901 #PZ0162 SIGMA, PLX4720 #S1152 SELLECKCHEM,, Autophagy inhibitors: 3-methyladenine (3-MA), #sc205596, Bafilomycin A1 #sc-201550 Santa Cruz Biotechnology, Inc.

    Techniques: Western Blot, Mutagenesis, Staining, Microscopy

    Physiological control of nuclear envelope blebbing. Frequencies of cNvj1p-EYFP–labeled blebs and vesicles as a function of growth, starvation, and rapamycin treatment in c NVJ1 ::EYFP YEF473a cells. (A) During growth in YPD (open circles, black line), cells at different OD 600 were scored for vacuole-associated nuclear envelope blebs and vesicles (squares, gray regression line). Cells in YPD were transferred to SD-N medium at different times during the growth curve, and after 3 h, were scored for blebs and vesicles (triangles, dashed regression line). (B) Cells grown in SCGlu were scored for blebs and vesicles (squares, gray regression line) as a function of the growth curve (circles, black line) and after treatment with rapamycin (gray triangles, dashed regression line) as described in MATERIALS AND METHODS. At least 100 cells were scored at each point.

    Journal: Molecular Biology of the Cell

    Article Title: Piecemeal Microautophagy of Nucleus in Saccharomyces cerevisiae

    doi: 10.1091/mbc.E02-08-0483

    Figure Lengend Snippet: Physiological control of nuclear envelope blebbing. Frequencies of cNvj1p-EYFP–labeled blebs and vesicles as a function of growth, starvation, and rapamycin treatment in c NVJ1 ::EYFP YEF473a cells. (A) During growth in YPD (open circles, black line), cells at different OD 600 were scored for vacuole-associated nuclear envelope blebs and vesicles (squares, gray regression line). Cells in YPD were transferred to SD-N medium at different times during the growth curve, and after 3 h, were scored for blebs and vesicles (triangles, dashed regression line). (B) Cells grown in SCGlu were scored for blebs and vesicles (squares, gray regression line) as a function of the growth curve (circles, black line) and after treatment with rapamycin (gray triangles, dashed regression line) as described in MATERIALS AND METHODS. At least 100 cells were scored at each point.

    Article Snippet: If cells were to be starved for nitrogen (SD-N) or glucose (SC-Glu) before imaging, they were first stained with FM4-64, and then washed 5× in SD-N or SC-Glu and resuspended in SD-N or SC-Glu media for 3 h. DNA was stained immediately before imaging with 5 μM Hoechst reagent H-1398 (Molecular Probes, Eugene, OR) in SCGlu or SD-N. For rapamycin treatment, cells in SCGlu were treated for 3 h with a final concentration of 0.2 μg/ml rapamycin by using a stock solution of 20 μg/ml rapamycin (Calbiochem, San Diego, CA) in 90% ethanol/10% Triton X-100.

    Techniques: Labeling

    Effect of rapamycin on GSPC self-renewal. A, rapamycin attenuated the efficiency of neurosphere formation of GSPCs. The percentage of neurosphere-forming cells and the total number of cells were determined. B, representative images of SU-2 neurospheres treated as indicated. The neurosphere is significantly smaller in the rapamycin group than in control group and 3-MA plus rapamycin group, suggesting that rapamycin reduced the self-renewal ability of GSPCs and that 3-MA blocked this effect.

    Journal: Chinese Journal of Cancer

    Article Title: Rapamycin induces differentiation of glioma stem/progenitor cells by activating autophagy

    doi: 10.5732/cjc.011.10234

    Figure Lengend Snippet: Effect of rapamycin on GSPC self-renewal. A, rapamycin attenuated the efficiency of neurosphere formation of GSPCs. The percentage of neurosphere-forming cells and the total number of cells were determined. B, representative images of SU-2 neurospheres treated as indicated. The neurosphere is significantly smaller in the rapamycin group than in control group and 3-MA plus rapamycin group, suggesting that rapamycin reduced the self-renewal ability of GSPCs and that 3-MA blocked this effect.

    Article Snippet: Rapamycin (cat #R0395) and 3-methyladenine (3-MA) were from Sigma.

    Techniques:

    Effect of rapamycin on autophagy of GSPCs in vivo . Representative electron micrograph images show increased autophagosomes following rapamycin treatment in an SU-2 xenograft model. Three mice from each group and ten fields for each mouse were examined and displayed similar morphologic changes. TEM showed intact nuclear membranes as well as normal chromatin structure and mitochondrial numbers and distribution in cases from the untreated group. TEM also showed a remarkable increase of double-membrane autophagosomes (as indicated by arrows) in the rapamycin group and in the 3-MA plus rapamycin group. N, nucleus.

    Journal: Chinese Journal of Cancer

    Article Title: Rapamycin induces differentiation of glioma stem/progenitor cells by activating autophagy

    doi: 10.5732/cjc.011.10234

    Figure Lengend Snippet: Effect of rapamycin on autophagy of GSPCs in vivo . Representative electron micrograph images show increased autophagosomes following rapamycin treatment in an SU-2 xenograft model. Three mice from each group and ten fields for each mouse were examined and displayed similar morphologic changes. TEM showed intact nuclear membranes as well as normal chromatin structure and mitochondrial numbers and distribution in cases from the untreated group. TEM also showed a remarkable increase of double-membrane autophagosomes (as indicated by arrows) in the rapamycin group and in the 3-MA plus rapamycin group. N, nucleus.

    Article Snippet: Rapamycin (cat #R0395) and 3-methyladenine (3-MA) were from Sigma.

    Techniques: In Vivo, Mouse Assay, Transmission Electron Microscopy

    Autophagy detection in glioma stem/progenitor cells (GSPCs) after treatment. SU-2 cells were treated with rapamycin, 3-methyladenine (3-MA) plus rapamycin, E64d plus rapamycin, or untreated as control. A, immunofluorescent microscopy shows changes in the localization of LC3 in GSPCs after indicated treatments. B, quantification of LC3 expression in GSPCs after indicated treatments. The positive rate of LC3 was significantly higher in cells treated with rapamycin alone or in combination with E64d than in untreated cells ( P

    Journal: Chinese Journal of Cancer

    Article Title: Rapamycin induces differentiation of glioma stem/progenitor cells by activating autophagy

    doi: 10.5732/cjc.011.10234

    Figure Lengend Snippet: Autophagy detection in glioma stem/progenitor cells (GSPCs) after treatment. SU-2 cells were treated with rapamycin, 3-methyladenine (3-MA) plus rapamycin, E64d plus rapamycin, or untreated as control. A, immunofluorescent microscopy shows changes in the localization of LC3 in GSPCs after indicated treatments. B, quantification of LC3 expression in GSPCs after indicated treatments. The positive rate of LC3 was significantly higher in cells treated with rapamycin alone or in combination with E64d than in untreated cells ( P

    Article Snippet: Rapamycin (cat #R0395) and 3-methyladenine (3-MA) were from Sigma.

    Techniques: Microscopy, Expressing

    Effect of rapamycin on GSPC differentiation in vivo . A, representative images of immunohistochemical staining of GFAP and Nestin in tumor sections at 35 days after indicated treatments. The expression of Nestin is weaker and the expression of GFAP is stronger in the rapamycin group than in the control and 3-MA plus rapamycin groups. B, positive rate of GFAP is significantly higher and that of Nestin is significantly lower in the rapamycin group than in the control and 3-MA plus rapamycin groups.

    Journal: Chinese Journal of Cancer

    Article Title: Rapamycin induces differentiation of glioma stem/progenitor cells by activating autophagy

    doi: 10.5732/cjc.011.10234

    Figure Lengend Snippet: Effect of rapamycin on GSPC differentiation in vivo . A, representative images of immunohistochemical staining of GFAP and Nestin in tumor sections at 35 days after indicated treatments. The expression of Nestin is weaker and the expression of GFAP is stronger in the rapamycin group than in the control and 3-MA plus rapamycin groups. B, positive rate of GFAP is significantly higher and that of Nestin is significantly lower in the rapamycin group than in the control and 3-MA plus rapamycin groups.

    Article Snippet: Rapamycin (cat #R0395) and 3-methyladenine (3-MA) were from Sigma.

    Techniques: In Vivo, Immunohistochemistry, Staining, Expressing

    Effect of rapamycin on GSPC differentiation. A, quantitative RT-PCR analysis was performed to determine the mRNA levels of the differentiation markers GFAP , Tuj1 , and 0lig2 in SU-2 neurospheres at 3 days after the indicated treatments, β-actin was used as an internal normalization control. Error bars represent mean ± SD. B, immunocytochemistry for the indicated proteins was performed in SU-2 neurospheres at 3 days after the indicated treatments on poly-L-lysine-coated coverslips. Nuclei were counterstained with DAPI.

    Journal: Chinese Journal of Cancer

    Article Title: Rapamycin induces differentiation of glioma stem/progenitor cells by activating autophagy

    doi: 10.5732/cjc.011.10234

    Figure Lengend Snippet: Effect of rapamycin on GSPC differentiation. A, quantitative RT-PCR analysis was performed to determine the mRNA levels of the differentiation markers GFAP , Tuj1 , and 0lig2 in SU-2 neurospheres at 3 days after the indicated treatments, β-actin was used as an internal normalization control. Error bars represent mean ± SD. B, immunocytochemistry for the indicated proteins was performed in SU-2 neurospheres at 3 days after the indicated treatments on poly-L-lysine-coated coverslips. Nuclei were counterstained with DAPI.

    Article Snippet: Rapamycin (cat #R0395) and 3-methyladenine (3-MA) were from Sigma.

    Techniques: Quantitative RT-PCR, Immunocytochemistry

    ArhGAP44 is recruited to collapsing artificial lamellipodia in neurons. ( A ) Rapamycin-induced dimerization assay. Neurons were quadruple-transfected with a membrane-anchored FRB, a fluorescently labeled FKBP that was associated with the Rac-GEF TIAM, a cytosolic reference, and the fluorescently labelled N-BAR domain of ArhGAP44. Addition of rapamycin triggered dimerization of FRB and FKBP, which led to a rapid translocation of TIAM to the plasma membrane. Enrichment of TIAM at the plasma membrane augmented local Rac activity and actin dynamics. ( B ) Neurons quadruple-transfected with the constructs before and after addition of rapamycin. Note the formation of ectopic actin-rich structures. ( C ) Time lapse images show enrichment of the N-BAR domain of ArhGAP44 at retracting actin-rich structures. Cells were quadruple-transfected with Lyn-FRB, CFP-FKBP-TIAM, YFP-ArhGAP44(N-BAR), and the empty mCherry plasmid as a cytosolic reference. Note the enrichment of the N-BAR domain of ArhGAP44 at the collapsing lamellipodia. ( D ) Kymograph of ArhGAP44(N-BAR) (top panel) and the cytosolic reference mCherry (middle panel) show relative enrichment of the N-BAR domain of ArhGAP44 at a retracting actin-rich structures (bottom panel). ( B ), 10 µm; ( C ), 2 µm. DOI: http://dx.doi.org/10.7554/eLife.03116.039

    Journal: eLife

    Article Title: Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons

    doi: 10.7554/eLife.03116

    Figure Lengend Snippet: ArhGAP44 is recruited to collapsing artificial lamellipodia in neurons. ( A ) Rapamycin-induced dimerization assay. Neurons were quadruple-transfected with a membrane-anchored FRB, a fluorescently labeled FKBP that was associated with the Rac-GEF TIAM, a cytosolic reference, and the fluorescently labelled N-BAR domain of ArhGAP44. Addition of rapamycin triggered dimerization of FRB and FKBP, which led to a rapid translocation of TIAM to the plasma membrane. Enrichment of TIAM at the plasma membrane augmented local Rac activity and actin dynamics. ( B ) Neurons quadruple-transfected with the constructs before and after addition of rapamycin. Note the formation of ectopic actin-rich structures. ( C ) Time lapse images show enrichment of the N-BAR domain of ArhGAP44 at retracting actin-rich structures. Cells were quadruple-transfected with Lyn-FRB, CFP-FKBP-TIAM, YFP-ArhGAP44(N-BAR), and the empty mCherry plasmid as a cytosolic reference. Note the enrichment of the N-BAR domain of ArhGAP44 at the collapsing lamellipodia. ( D ) Kymograph of ArhGAP44(N-BAR) (top panel) and the cytosolic reference mCherry (middle panel) show relative enrichment of the N-BAR domain of ArhGAP44 at a retracting actin-rich structures (bottom panel). ( B ), 10 µm; ( C ), 2 µm. DOI: http://dx.doi.org/10.7554/eLife.03116.039

    Article Snippet: 24 hr later, individual cells were imaged before and after addition of 100 nM rapamycin (B0560; Sigma–Aldrich) with a 63× objective and a 1.5× Optovar module.

    Techniques: Transfection, Labeling, Translocation Assay, Activity Assay, Construct, Plasmid Preparation

    Evident Autophagy Was Observed in Iso-GNA Treated 549 cells. (A) LC3 expression in A549 cells. Different concentrations of iso-GNA were incubated with A549 cells for 24 h. and 3-MA (2 mM) was treated as an autophagy inhibitor. (B) SQSTM1(p62) expression in A549 cells. Different concentrations of iso-GNA were incubated with A549 cells for 24 h. (C) 10 μM iso-GNA were treated with A549 cells(expression RFP-LC3)for 24 h. RFP-LC3 dots was observed by Leica aser-scanning spectrum confocal system. (D) 10 μM iso-GNA were treated with A549 cells for 24 h, then cells were subjected to transmission electron microscopy to observe double-membraned vacuolation (arrows) (E) Various concentrations of iso-GNA were treated with A549 cells for 24 h. then Beclin 1, Atg7 and Atg12-Atg5 complex expression level were detected by western blot. (F) A549 cells treated with 2 μM rapamycin, 10 μM iso-GNA with or with out 4 mM 3-MA for 24 h. Then LC3 immunofluorescence image in A549 cells were detected by a fluorescence microscope. Data of three independent tests were shown as means ± s.d. *p

    Journal: Scientific Reports

    Article Title: Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

    doi: 10.1038/srep07697

    Figure Lengend Snippet: Evident Autophagy Was Observed in Iso-GNA Treated 549 cells. (A) LC3 expression in A549 cells. Different concentrations of iso-GNA were incubated with A549 cells for 24 h. and 3-MA (2 mM) was treated as an autophagy inhibitor. (B) SQSTM1(p62) expression in A549 cells. Different concentrations of iso-GNA were incubated with A549 cells for 24 h. (C) 10 μM iso-GNA were treated with A549 cells(expression RFP-LC3)for 24 h. RFP-LC3 dots was observed by Leica aser-scanning spectrum confocal system. (D) 10 μM iso-GNA were treated with A549 cells for 24 h, then cells were subjected to transmission electron microscopy to observe double-membraned vacuolation (arrows) (E) Various concentrations of iso-GNA were treated with A549 cells for 24 h. then Beclin 1, Atg7 and Atg12-Atg5 complex expression level were detected by western blot. (F) A549 cells treated with 2 μM rapamycin, 10 μM iso-GNA with or with out 4 mM 3-MA for 24 h. Then LC3 immunofluorescence image in A549 cells were detected by a fluorescence microscope. Data of three independent tests were shown as means ± s.d. *p

    Article Snippet: DMSO, MTT, cisplatin, bafilomycin A1, chloroquine, zVAD.fmk, 3-MA, DAPI and rapamycin were obtained from Sigma Aldrich.

    Techniques: Expressing, Incubation, Transmission Assay, Electron Microscopy, Western Blot, Immunofluorescence, Fluorescence, Microscopy

    Iso-GNA induced autophagic cell death through the inhibition of Akt/mTOR Pathway. (A) Western blot was used to detect the p-Akt, Akt, p-mTOR, mTOR, p-p70 S6K, p70 S6K, p-4E-BP1 and 4E-BP1 expression in various concentrations of iso-GNA treated A549 cells. (B) 0, 10 μM of iso-GNA with or without 1 μM rapamycin were added to A549 cells for 24 h, western blot was employed to detect the LC3 expression level. (C) The indicated concentration of iso-GNA with or without 1 μM rapamycin were treated with A549 cells for 24 h, trypan blue dye was employed to stain the cells and analyze the death ratio. Data of three independent tests were shown as means ± s.d. *p

    Journal: Scientific Reports

    Article Title: Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

    doi: 10.1038/srep07697

    Figure Lengend Snippet: Iso-GNA induced autophagic cell death through the inhibition of Akt/mTOR Pathway. (A) Western blot was used to detect the p-Akt, Akt, p-mTOR, mTOR, p-p70 S6K, p70 S6K, p-4E-BP1 and 4E-BP1 expression in various concentrations of iso-GNA treated A549 cells. (B) 0, 10 μM of iso-GNA with or without 1 μM rapamycin were added to A549 cells for 24 h, western blot was employed to detect the LC3 expression level. (C) The indicated concentration of iso-GNA with or without 1 μM rapamycin were treated with A549 cells for 24 h, trypan blue dye was employed to stain the cells and analyze the death ratio. Data of three independent tests were shown as means ± s.d. *p

    Article Snippet: DMSO, MTT, cisplatin, bafilomycin A1, chloroquine, zVAD.fmk, 3-MA, DAPI and rapamycin were obtained from Sigma Aldrich.

    Techniques: Inhibition, Western Blot, Expressing, Concentration Assay, Staining

    AIPD as a tool for quantifying autophagic flux. ( A ) Overlaps of the 2 channels of mRFP-EGFP-LC3B for control (CTRL), serum-starved (STARV), rapamycin treated (RAP), rapamycin-treated and serum-starved cells (STARV + RAP), chloroquine-treated (CHL) and 3-MA-treated cells (3-MA). ( B ) R-maps of the cells with the above-mentioned treatments (16-color rainbow lookup table [ImageJ]), values from R 0 -black to R f -white. ( C ) AIPDs for the cells with the above-mentioned treatments. Scale bar: 20 µm. A 3-Lorentzian deconvolution of the AIPD function allows measuring the relative contributions of the 3 populations: the first population represents autophagosomes (AP, pH = 6.0, orange line), whether the second and the third represent 2 different states of autolysosomes (AL), an early one (AL E , pH = 5.6, green line), and a mature, more acidic state (AL M , pH = 5.2, blue line). The black line represents the sum of the 3 populations.

    Journal: Autophagy

    Article Title: Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates

    doi: 10.1080/15548627.2015.1084455

    Figure Lengend Snippet: AIPD as a tool for quantifying autophagic flux. ( A ) Overlaps of the 2 channels of mRFP-EGFP-LC3B for control (CTRL), serum-starved (STARV), rapamycin treated (RAP), rapamycin-treated and serum-starved cells (STARV + RAP), chloroquine-treated (CHL) and 3-MA-treated cells (3-MA). ( B ) R-maps of the cells with the above-mentioned treatments (16-color rainbow lookup table [ImageJ]), values from R 0 -black to R f -white. ( C ) AIPDs for the cells with the above-mentioned treatments. Scale bar: 20 µm. A 3-Lorentzian deconvolution of the AIPD function allows measuring the relative contributions of the 3 populations: the first population represents autophagosomes (AP, pH = 6.0, orange line), whether the second and the third represent 2 different states of autolysosomes (AL), an early one (AL E , pH = 5.6, green line), and a mature, more acidic state (AL M , pH = 5.2, blue line). The black line represents the sum of the 3 populations.

    Article Snippet: Cells were left to adhere and spread for 4.5 h, then the medium was replaced with serum-free Dulbecco's modified Eagle's medium, and 5% fetal calf serum, 200 nM rapamycin (LC Laboratorier, R-5000) or 100 µM chloroquine (Sigma, C6628) added alone or in combination where required.

    Techniques:

    pH and absolute number of autophagic intermediates. ( A ) R-map (

    Journal: Autophagy

    Article Title: Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates

    doi: 10.1080/15548627.2015.1084455

    Figure Lengend Snippet: pH and absolute number of autophagic intermediates. ( A ) R-map ("cool" lookup table [imageJ], values from R 0 -black to R f -white) of LysoSensor-marked cells of the control, serum-starved, rapamycin-treated, rapamycin+starvation-treated, chloroquine-treated and 3-MA-treated samples. ( B ) LPD of ctrl untreated cells serum-starved, rapamycin-treated, rapamycin+starvation-treated, chloroquine-treated, and 3-MA-treated samples. Scale bar: 20 µm. A 2-Lorentzian deconvolution of the AIPD function can be recovered to distinguish between the number of acidic organelles as lysosomes (L, pink line) and autophagic intermediates (AI = AP+AL, purple line). The black line is the sum of the 2 Lorentzians. ( C ) pH, weighted by the LPD distributions, for lysosomes (L, pH = 5.0, pink line), and autophagic intermediates (AI, pH = 5.4, purple line). ( D ) Stacked column histogram of the number of puncta per cell for L (pink) and AI (purple), calculated from LPD area fractions. Error bars represent the error associated to the fitting algorithm.

    Article Snippet: Cells were left to adhere and spread for 4.5 h, then the medium was replaced with serum-free Dulbecco's modified Eagle's medium, and 5% fetal calf serum, 200 nM rapamycin (LC Laboratorier, R-5000) or 100 µM chloroquine (Sigma, C6628) added alone or in combination where required.

    Techniques:

    HCMV-induced metabolic reprogramming of amino acid metabolism. (A- C) MRC5 cells were mock or HCMV-infected (MOI = 3). At 36hpi, cellular medium was renewed, harvested 24h later (60hpi), and analyzed for changes in metabolite levels. Values are means ± SE (n = 6). (D, E, G H) MRC5 cells were infected as in (A-C). At 36hpi, fresh medium containing DMSO (+DMSO) or 100 nm of rapamycin (+Rap) was added to the plates and conditioned medium and cells were harvested after 24h (60hpi). (D) Western blot analysis of drug treated mock or HCMV-infected cells (E, G H) Changes in metabolic intermediates present in the conditioned medium were measured. Values are means ± SE (n = 3) (*p

    Journal: PLoS Pathogens

    Article Title: The Human Cytomegalovirus UL38 protein drives mTOR-independent metabolic flux reprogramming by inhibiting TSC2

    doi: 10.1371/journal.ppat.1007569

    Figure Lengend Snippet: HCMV-induced metabolic reprogramming of amino acid metabolism. (A- C) MRC5 cells were mock or HCMV-infected (MOI = 3). At 36hpi, cellular medium was renewed, harvested 24h later (60hpi), and analyzed for changes in metabolite levels. Values are means ± SE (n = 6). (D, E, G H) MRC5 cells were infected as in (A-C). At 36hpi, fresh medium containing DMSO (+DMSO) or 100 nm of rapamycin (+Rap) was added to the plates and conditioned medium and cells were harvested after 24h (60hpi). (D) Western blot analysis of drug treated mock or HCMV-infected cells (E, G H) Changes in metabolic intermediates present in the conditioned medium were measured. Values are means ± SE (n = 3) (*p

    Article Snippet: Compounds Rapamycin (Sigma-Aldrich) and Torin-1 (ApexBio) were prepared at 100uM and 250uM respectively in dimethyl sulfoxide (DMSO).

    Techniques: Infection, Western Blot

    TSC2 knock-down induces activation of metabolic fluxes independent of mTORC1. (A-D) HFF cells were transduced with control (pLKO) or TSC2-specific shRNA (TSC2 KD) -expressing lentiviruses and selected. Confluent cells were cultured in serum free media for 24h, at which time the conditioned medium and cells were harvested for analysis. (A) Western blot analysis of pLKO and TSC2 KD cells. Protein band intensities are shown relative to the pLKO control value. Arrows indicate both p70 and p85 isoform of S6K (B-D). Changes in metabolic intermediates present in the conditioned medium were measured. Values are means ± SE (n = 4). (E-H) Confluent HFF cells expressing pLKO or TSC2-specific shRNA were cultured in serum free media containing DMSO (+DMSO) or 100 nm of rapamycin (+Rap) for 24h, at which time the conditioned medium and cells were harvested for analysis. (E) Western blot analysis of pLKO and TSC2 KD drug treated cells. Protein band intensities are shown relative to the pLKO+DMSO control value. (F-H) Changes in metabolic intermediates present in the conditioned medium were measured by LC-MS/MS. Values are means ± SE (n = 4). (*p

    Journal: PLoS Pathogens

    Article Title: The Human Cytomegalovirus UL38 protein drives mTOR-independent metabolic flux reprogramming by inhibiting TSC2

    doi: 10.1371/journal.ppat.1007569

    Figure Lengend Snippet: TSC2 knock-down induces activation of metabolic fluxes independent of mTORC1. (A-D) HFF cells were transduced with control (pLKO) or TSC2-specific shRNA (TSC2 KD) -expressing lentiviruses and selected. Confluent cells were cultured in serum free media for 24h, at which time the conditioned medium and cells were harvested for analysis. (A) Western blot analysis of pLKO and TSC2 KD cells. Protein band intensities are shown relative to the pLKO control value. Arrows indicate both p70 and p85 isoform of S6K (B-D). Changes in metabolic intermediates present in the conditioned medium were measured. Values are means ± SE (n = 4). (E-H) Confluent HFF cells expressing pLKO or TSC2-specific shRNA were cultured in serum free media containing DMSO (+DMSO) or 100 nm of rapamycin (+Rap) for 24h, at which time the conditioned medium and cells were harvested for analysis. (E) Western blot analysis of pLKO and TSC2 KD drug treated cells. Protein band intensities are shown relative to the pLKO+DMSO control value. (F-H) Changes in metabolic intermediates present in the conditioned medium were measured by LC-MS/MS. Values are means ± SE (n = 4). (*p

    Article Snippet: Compounds Rapamycin (Sigma-Aldrich) and Torin-1 (ApexBio) were prepared at 100uM and 250uM respectively in dimethyl sulfoxide (DMSO).

    Techniques: Activation Assay, Transduction, shRNA, Expressing, Cell Culture, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    U L 38-induced metabolic flux activation is mTOR independent. After 24h incubation, conditioned serum free medium from confluent MRC5 cells transduced with an empty vector or U L 38 was harvested for analysis. Media contained DMSO (+DMSO), 100 nm of rapamycin (+Rap) or 250nM of Torin-1 (TOR or +Torin1) as indicated. (A, E) Western blot analysis of treated EV and U L 38 cells. (B, C, D, F G) Changes in metabolic intermediates present in the conditioned medium were measured. Values are means ± SE (B-D n = 9, F-G n = 8). (*p

    Journal: PLoS Pathogens

    Article Title: The Human Cytomegalovirus UL38 protein drives mTOR-independent metabolic flux reprogramming by inhibiting TSC2

    doi: 10.1371/journal.ppat.1007569

    Figure Lengend Snippet: U L 38-induced metabolic flux activation is mTOR independent. After 24h incubation, conditioned serum free medium from confluent MRC5 cells transduced with an empty vector or U L 38 was harvested for analysis. Media contained DMSO (+DMSO), 100 nm of rapamycin (+Rap) or 250nM of Torin-1 (TOR or +Torin1) as indicated. (A, E) Western blot analysis of treated EV and U L 38 cells. (B, C, D, F G) Changes in metabolic intermediates present in the conditioned medium were measured. Values are means ± SE (B-D n = 9, F-G n = 8). (*p

    Article Snippet: Compounds Rapamycin (Sigma-Aldrich) and Torin-1 (ApexBio) were prepared at 100uM and 250uM respectively in dimethyl sulfoxide (DMSO).

    Techniques: Activation Assay, Incubation, Transduction, Plasmid Preparation, Western Blot

    Induction of phenotypic changes in MCF-7 cells treated with CN-A and rapamycin. Upper row: E-cadherin. Cells were cultured in the presence (right) or absence (left) of 10 μg/ml CN-A plus 0.5 ng/ml of rapamycin for 7 days. Lower row: SA-βGal activity. Cells were cultured in the presence (right) or absence (left) of 10 μg/ml CN-A plus 0.5 ng/ml rapamycin for 7 days. CN-A, cotylenin A; Rapa, rapamycin; SA-βGal, senescence-associated β-galactosidase.

    Journal: Breast Cancer Research

    Article Title: Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

    doi: 10.1186/bcr1344

    Figure Lengend Snippet: Induction of phenotypic changes in MCF-7 cells treated with CN-A and rapamycin. Upper row: E-cadherin. Cells were cultured in the presence (right) or absence (left) of 10 μg/ml CN-A plus 0.5 ng/ml of rapamycin for 7 days. Lower row: SA-βGal activity. Cells were cultured in the presence (right) or absence (left) of 10 μg/ml CN-A plus 0.5 ng/ml rapamycin for 7 days. CN-A, cotylenin A; Rapa, rapamycin; SA-βGal, senescence-associated β-galactosidase.

    Article Snippet: Materials Rapamycin was purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Cell Culture, Activity Assay

    Effects of rapamycin and CN-A on the growth of MCF-7 cells as xenografts. Mice were given a daily intraperitoneal injection of 100 ng rapamycin (black square, black circle) and/or subcutaneous injection of 100 μg of CN-A (black triangle, black circle) every other day. Values are expressed as mean ± standard deviation of 20 mice. Black diamond, untreated mice; white circle, mice treated with solvent alone. CN-A, cotylenin A; *, p

    Journal: Breast Cancer Research

    Article Title: Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

    doi: 10.1186/bcr1344

    Figure Lengend Snippet: Effects of rapamycin and CN-A on the growth of MCF-7 cells as xenografts. Mice were given a daily intraperitoneal injection of 100 ng rapamycin (black square, black circle) and/or subcutaneous injection of 100 μg of CN-A (black triangle, black circle) every other day. Values are expressed as mean ± standard deviation of 20 mice. Black diamond, untreated mice; white circle, mice treated with solvent alone. CN-A, cotylenin A; *, p

    Article Snippet: Materials Rapamycin was purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Mouse Assay, Injection, Standard Deviation

    Time courses of TGFBI, BIK, cyclin G 2 and GRB7 mRNA expression in MCF-7 cells. (a) MCF-7 cells were treated without (lanes 1, 5, 9, 13 and 17) or with 0.5 ng/ml rapamycin (lanes 2, 6, 10, 14 and 18), 10 μg/ml CN-A (lanes 3, 7, 11, 15 and 19), or 0.5 ng/ml rapamycin plus 10 μg/ml CN-A (lanes 4, 8, 12, 16 and 20) for 1, 3, 6, 12 and 24 hours. Expression of mRNAs was examined by RT-PCR analysis. (b) Graphs depict the relative levels of mRNA after normalization to GAPDH mRNA levels in Fig. 5a. Cells were treated with 0.5 ng/ml rapamycin (white triangle) or 10 μg/ml CN-A (white square), or both combined (black circle). BIK, BCL2-interacting killer; CN-A, cotylenin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRB, growth factor receptor-bound; TGFBI, transforming growth factor-β-induced 68 kDa protein.

    Journal: Breast Cancer Research

    Article Title: Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

    doi: 10.1186/bcr1344

    Figure Lengend Snippet: Time courses of TGFBI, BIK, cyclin G 2 and GRB7 mRNA expression in MCF-7 cells. (a) MCF-7 cells were treated without (lanes 1, 5, 9, 13 and 17) or with 0.5 ng/ml rapamycin (lanes 2, 6, 10, 14 and 18), 10 μg/ml CN-A (lanes 3, 7, 11, 15 and 19), or 0.5 ng/ml rapamycin plus 10 μg/ml CN-A (lanes 4, 8, 12, 16 and 20) for 1, 3, 6, 12 and 24 hours. Expression of mRNAs was examined by RT-PCR analysis. (b) Graphs depict the relative levels of mRNA after normalization to GAPDH mRNA levels in Fig. 5a. Cells were treated with 0.5 ng/ml rapamycin (white triangle) or 10 μg/ml CN-A (white square), or both combined (black circle). BIK, BCL2-interacting killer; CN-A, cotylenin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRB, growth factor receptor-bound; TGFBI, transforming growth factor-β-induced 68 kDa protein.

    Article Snippet: Materials Rapamycin was purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Classic isobologram at IC 50 : rapamycin plus CN-A in MCF-7 cells. Isoboles for the combination of rapamycin with CN-A that were isoeffective (IC 50 ) for inhibition of proliferation of MCF-7 cells. The dashed line indicates the zero interaction of the isobole. Cells (3 × 10 4 cells/ml) were treated with rapamycin and CN-A for 5 days. Values are the means of three separate experiments. CN-A, cotylenin A; IC 50 , the concentration of the drug required for 50 % inhibition of cell growth.

    Journal: Breast Cancer Research

    Article Title: Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

    doi: 10.1186/bcr1344

    Figure Lengend Snippet: Classic isobologram at IC 50 : rapamycin plus CN-A in MCF-7 cells. Isoboles for the combination of rapamycin with CN-A that were isoeffective (IC 50 ) for inhibition of proliferation of MCF-7 cells. The dashed line indicates the zero interaction of the isobole. Cells (3 × 10 4 cells/ml) were treated with rapamycin and CN-A for 5 days. Values are the means of three separate experiments. CN-A, cotylenin A; IC 50 , the concentration of the drug required for 50 % inhibition of cell growth.

    Article Snippet: Materials Rapamycin was purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Inhibition, Concentration Assay

    Combined effect of rapamycin and CN-A on the growth of human cancer cells. (a) human breast cancer T-47D cells (3 × 10 4 cells/ml) were treated with CN-A in the absence (black triangle) or the presence (black circle) of 0.5 ng/ml rapamycin for 5 days. (b) human breast cancer MBA-MB-231 (3 × 10 4 cells/ml) cells were treated with CN-A in the absence (black triangle) or the presence (black circle) of 15 ng/ml rapamycin for 5 days. (c) human promyelocytic leukaemia NB-4 cells (3 × 10 4 cells/ml) were treated with CN-A in the absence (black triangle) or the presence (black circle) of 0.5 ng/ml rapamycin for 5 days. Values are expressed as mean ± standard deviation for three separate experiments. CN-A, cotylenin A.

    Journal: Breast Cancer Research

    Article Title: Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

    doi: 10.1186/bcr1344

    Figure Lengend Snippet: Combined effect of rapamycin and CN-A on the growth of human cancer cells. (a) human breast cancer T-47D cells (3 × 10 4 cells/ml) were treated with CN-A in the absence (black triangle) or the presence (black circle) of 0.5 ng/ml rapamycin for 5 days. (b) human breast cancer MBA-MB-231 (3 × 10 4 cells/ml) cells were treated with CN-A in the absence (black triangle) or the presence (black circle) of 15 ng/ml rapamycin for 5 days. (c) human promyelocytic leukaemia NB-4 cells (3 × 10 4 cells/ml) were treated with CN-A in the absence (black triangle) or the presence (black circle) of 0.5 ng/ml rapamycin for 5 days. Values are expressed as mean ± standard deviation for three separate experiments. CN-A, cotylenin A.

    Article Snippet: Materials Rapamycin was purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Standard Deviation

    Induction of G 1 arrest in MCF-7 cells treated with rapamycin plus CN-A. Cells were cultured (a) without or (b) with 10 μg/ml CN-A, (c) 0.5 ng/ml rapamycin, or (d) 0.5 ng/ml rapamycin plus 10 μg/ml CN-A for 6 days, and DNA histograms were then analyzed. CN-A, cotylenin A.

    Journal: Breast Cancer Research

    Article Title: Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

    doi: 10.1186/bcr1344

    Figure Lengend Snippet: Induction of G 1 arrest in MCF-7 cells treated with rapamycin plus CN-A. Cells were cultured (a) without or (b) with 10 μg/ml CN-A, (c) 0.5 ng/ml rapamycin, or (d) 0.5 ng/ml rapamycin plus 10 μg/ml CN-A for 6 days, and DNA histograms were then analyzed. CN-A, cotylenin A.

    Article Snippet: Materials Rapamycin was purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Cell Culture

    Effects of rapamycin and CN-A on the expressions of p53, p21 Cip1 , p27 Kip1 and cyclins. Expression of mRNAs was examined by RT-PCR analysis. (a) MCF-7 cells were cultured with 2.5–10 μg/ml CN-A or 1 ng/ml rapamycin, or both combined for 24 hours. (b) MCF-7 cells were cultured with 0.5 ng/ml rapamycin or 10 μg/ml CN-A, or both combined for 12 hours. The levels of GAPDH expression are shown to demonstrate that equal amounts of RNA were used for RT-PCR. CN-A, cotylenin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Journal: Breast Cancer Research

    Article Title: Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

    doi: 10.1186/bcr1344

    Figure Lengend Snippet: Effects of rapamycin and CN-A on the expressions of p53, p21 Cip1 , p27 Kip1 and cyclins. Expression of mRNAs was examined by RT-PCR analysis. (a) MCF-7 cells were cultured with 2.5–10 μg/ml CN-A or 1 ng/ml rapamycin, or both combined for 24 hours. (b) MCF-7 cells were cultured with 0.5 ng/ml rapamycin or 10 μg/ml CN-A, or both combined for 12 hours. The levels of GAPDH expression are shown to demonstrate that equal amounts of RNA were used for RT-PCR. CN-A, cotylenin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Article Snippet: Materials Rapamycin was purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    Synergistic effects of rapamycin and CN-A on the growth of MCF-7 cells. (a) Cells (3 × 10 4 cells/ml) were cultured with various concentrations of rapamycin and CN-A for 5 days. After culture, cells were stained with May–Grunwald–Giemsa. (b) Cells (3 × 10 4 cells/ml) were cultured without (white circle) or with (white triangle) 0.5 ng/ml rapamycin, 10 μg/ml CN-A (white square), or 0.5 ng/ml rapamycin plus 10 μg/ml CN-A (black circle) for the indicated number of days. The values are expressed as mean ± standard deviation of four determinations. (c) Cells were cultured without (white circle) or with (white triangle) 0.5 ng/ml rapamycin, 10 μg/ml CN-A (white square), or 0.5 ng/ml rapamycin plus 10 μg/ml CN-A (black circle) for the indicated number of days. The culture medium was replaced by fresh medium at least once every 5 days. The cell density was kept at 1–8 × 10 4 cells/ml. The values are expressed as mean ± SD of four separate experiments. CN-A, cotylenin A.

    Journal: Breast Cancer Research

    Article Title: Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

    doi: 10.1186/bcr1344

    Figure Lengend Snippet: Synergistic effects of rapamycin and CN-A on the growth of MCF-7 cells. (a) Cells (3 × 10 4 cells/ml) were cultured with various concentrations of rapamycin and CN-A for 5 days. After culture, cells were stained with May–Grunwald–Giemsa. (b) Cells (3 × 10 4 cells/ml) were cultured without (white circle) or with (white triangle) 0.5 ng/ml rapamycin, 10 μg/ml CN-A (white square), or 0.5 ng/ml rapamycin plus 10 μg/ml CN-A (black circle) for the indicated number of days. The values are expressed as mean ± standard deviation of four determinations. (c) Cells were cultured without (white circle) or with (white triangle) 0.5 ng/ml rapamycin, 10 μg/ml CN-A (white square), or 0.5 ng/ml rapamycin plus 10 μg/ml CN-A (black circle) for the indicated number of days. The culture medium was replaced by fresh medium at least once every 5 days. The cell density was kept at 1–8 × 10 4 cells/ml. The values are expressed as mean ± SD of four separate experiments. CN-A, cotylenin A.

    Article Snippet: Materials Rapamycin was purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Cell Culture, Staining, Standard Deviation

    The effects of the 9E4 monoclonal antibody on promotion of α-syn clearance via autophagy in a neuronal cell model. (A) Baseline co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-control and treated with the IgG1 control antibody. (B) Baseline co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn and treated with the IgG1 control antibody. (C) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn and treated with the 9E4 antibody. (D) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-control, treated with the IgG1 control antibody and rapamycin, an inducer of autophagy. (E) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin, an inducer of autophagy. (F) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA, an inhibitor of autophagy. (G) Analysis of α-syn immunoreactivity in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin. (H) Analysis of LC3-GFP signal in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin. (I) Analysis of α-syn immunoreactivity in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA. (J) Quantitative analysis of LC3-GFP signal in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA. Scale bar (A–F) = 10 µM (*) Indicates p

    Journal: PLoS ONE

    Article Title: Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease

    doi: 10.1371/journal.pone.0019338

    Figure Lengend Snippet: The effects of the 9E4 monoclonal antibody on promotion of α-syn clearance via autophagy in a neuronal cell model. (A) Baseline co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-control and treated with the IgG1 control antibody. (B) Baseline co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn and treated with the IgG1 control antibody. (C) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn and treated with the 9E4 antibody. (D) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-control, treated with the IgG1 control antibody and rapamycin, an inducer of autophagy. (E) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin, an inducer of autophagy. (F) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA, an inhibitor of autophagy. (G) Analysis of α-syn immunoreactivity in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin. (H) Analysis of LC3-GFP signal in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin. (I) Analysis of α-syn immunoreactivity in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA. (J) Quantitative analysis of LC3-GFP signal in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA. Scale bar (A–F) = 10 µM (*) Indicates p

    Article Snippet: Five hours after plating, cells were infected with the LV-αsyn LV-control and incubated for 24 hours with 9E4 (3 µg/ml) or IgG1 control in the presence or absence of inhibitors of the autophagy pathway – 3-methyladenine (3-MA, 10 mM, Sigma) or inducers – rapamycin (200 nM, Sigma) as previously described .

    Techniques: Infection

    Fluvastatin inhibits TF expression in SMC. In human saphenous vein SMC (HSVSMC, panel A, n = 4) as well as in human aortic SMC (HAoSMC, panel B, n = 4) sirolimus (20 ng/ml, 24 hours) up-regulated TF expression which was significantly inhibited by fluvastatin (1 μmol/L). Basal activity of RhoA was not influenced by sirolimus (20 ng/ml, n = 3, panel C), while the basal activity of mTOR was fully inhibited by sirolimus (20 ng/ml, 1 hour, n = 3, panel D). * = p

    Journal: BMC Cardiovascular Disorders

    Article Title: Sirolimus increases tissue factor expression but not activity in cultured human vascular smooth muscle cells

    doi: 10.1186/1471-2261-5-22

    Figure Lengend Snippet: Fluvastatin inhibits TF expression in SMC. In human saphenous vein SMC (HSVSMC, panel A, n = 4) as well as in human aortic SMC (HAoSMC, panel B, n = 4) sirolimus (20 ng/ml, 24 hours) up-regulated TF expression which was significantly inhibited by fluvastatin (1 μmol/L). Basal activity of RhoA was not influenced by sirolimus (20 ng/ml, n = 3, panel C), while the basal activity of mTOR was fully inhibited by sirolimus (20 ng/ml, 1 hour, n = 3, panel D). * = p

    Article Snippet: Materials Sirolimus was purchased from Calbiochem (Lucerne, Switzerland); fluvastatin was kindly provided by Novartis (Basel, Switzerland); tumor necrosis factor-α (TNF-α) was purchased from R & D, France); monoclonal mouse anti-TF antibody and tissue factor activity kit were purchased from American Diagnostica Inc (Socochim, Lausanne, Switzerland); anti-tubulin and all the other chemicals for immunoblotting were purchased from Sigma (Buchs, Switzerland); anti-phospho p70s6k (T389) was from Cell Signaling Technology.

    Techniques: Expressing, Activity Assay

    Sirolimus up-regulates tissue factor (TF) expression in SMC. (A). Sirolimus enhances TF expression in a concentration- and (B) time-dependent manner in human SMC. n = 6, * = p

    Journal: BMC Cardiovascular Disorders

    Article Title: Sirolimus increases tissue factor expression but not activity in cultured human vascular smooth muscle cells

    doi: 10.1186/1471-2261-5-22

    Figure Lengend Snippet: Sirolimus up-regulates tissue factor (TF) expression in SMC. (A). Sirolimus enhances TF expression in a concentration- and (B) time-dependent manner in human SMC. n = 6, * = p

    Article Snippet: Materials Sirolimus was purchased from Calbiochem (Lucerne, Switzerland); fluvastatin was kindly provided by Novartis (Basel, Switzerland); tumor necrosis factor-α (TNF-α) was purchased from R & D, France); monoclonal mouse anti-TF antibody and tissue factor activity kit were purchased from American Diagnostica Inc (Socochim, Lausanne, Switzerland); anti-tubulin and all the other chemicals for immunoblotting were purchased from Sigma (Buchs, Switzerland); anti-phospho p70s6k (T389) was from Cell Signaling Technology.

    Techniques: Expressing, Concentration Assay

    Sirolimus does not stimulate TF release and activity from SMC. Human SMC released much higher TF activity into the culture medium than endothelial cells (HUVECs) (p

    Journal: BMC Cardiovascular Disorders

    Article Title: Sirolimus increases tissue factor expression but not activity in cultured human vascular smooth muscle cells

    doi: 10.1186/1471-2261-5-22

    Figure Lengend Snippet: Sirolimus does not stimulate TF release and activity from SMC. Human SMC released much higher TF activity into the culture medium than endothelial cells (HUVECs) (p

    Article Snippet: Materials Sirolimus was purchased from Calbiochem (Lucerne, Switzerland); fluvastatin was kindly provided by Novartis (Basel, Switzerland); tumor necrosis factor-α (TNF-α) was purchased from R & D, France); monoclonal mouse anti-TF antibody and tissue factor activity kit were purchased from American Diagnostica Inc (Socochim, Lausanne, Switzerland); anti-tubulin and all the other chemicals for immunoblotting were purchased from Sigma (Buchs, Switzerland); anti-phospho p70s6k (T389) was from Cell Signaling Technology.

    Techniques: Activity Assay

    Rapamycin treatment rescues the ciliary length phenotype of Tsc1 − / − cells. a IFM analysis of the expression of the proliferation marker KI67 and formation of primary cilia (arrows), labeled with anti-acetylated α-tubulin (Ac-TUB) antibody, in Tsc1 − / − MEFs cultured in normal serum medium (left panels), in starvation medium (0.5% FBS) for 48 h (middle panels, with few proliferating cells), or after 48 h starvation followed by serum re-stimulation for 24 h (right panels, with many proliferating cells). b WB analysis of WT MEFs subjected to siRNA-mediated knock down of Tsc1 or Tsc2 . WT MEFs were analyzed 48 h after transfection with siTsc1, siTsc2 or siScramble (negative control). GAPDH was used as loading control. c IFM analysis of primary cilia in WT MEFs after knock down of Tsc1 or Tsc2. Cilia (arrows) were labeled with Ac-TUB antibodies and the ciliary base/centrosomes (asterisks) were labeled with anti-γ-tubulin antibody (γ-TUB). Nuclei were visualized with DAPI staining. Scale bar: 5 µM. d Quantification of ciliary lengths for experiment shown in c . Three hundred cilia from three independent experiments were used for quantification; error bars represent SEM. e IFM analysis of primary cilia (Ac-TUB) in Tsc1 − / − and Tsc2 − / − MEFs subjected to siRNA-mediated knock down followed by culturing in starvation medium (0.5% serum) for 48 h (24 h after transfection) to induce cilia formation. Nuclei were visualized with DAPI staining. Scale bar: 5 µM. f Quantification of ciliary lengths for experiment shown in e . One hundred cilia from three independent experiments were used for quantification; error bars represent SEM. g Quantification of ciliary lengths labeled with Ac-TUB antibody in WT, Tsc1 − / − and Tsc2 − / − MEFs cultured in low 48 h in the presence or absence of rapamycin

    Journal: Cellular and Molecular Life Sciences

    Article Title: TSC1 and TSC2 regulate cilia length and canonical Hedgehog signaling via different mechanisms

    doi: 10.1007/s00018-018-2761-8

    Figure Lengend Snippet: Rapamycin treatment rescues the ciliary length phenotype of Tsc1 − / − cells. a IFM analysis of the expression of the proliferation marker KI67 and formation of primary cilia (arrows), labeled with anti-acetylated α-tubulin (Ac-TUB) antibody, in Tsc1 − / − MEFs cultured in normal serum medium (left panels), in starvation medium (0.5% FBS) for 48 h (middle panels, with few proliferating cells), or after 48 h starvation followed by serum re-stimulation for 24 h (right panels, with many proliferating cells). b WB analysis of WT MEFs subjected to siRNA-mediated knock down of Tsc1 or Tsc2 . WT MEFs were analyzed 48 h after transfection with siTsc1, siTsc2 or siScramble (negative control). GAPDH was used as loading control. c IFM analysis of primary cilia in WT MEFs after knock down of Tsc1 or Tsc2. Cilia (arrows) were labeled with Ac-TUB antibodies and the ciliary base/centrosomes (asterisks) were labeled with anti-γ-tubulin antibody (γ-TUB). Nuclei were visualized with DAPI staining. Scale bar: 5 µM. d Quantification of ciliary lengths for experiment shown in c . Three hundred cilia from three independent experiments were used for quantification; error bars represent SEM. e IFM analysis of primary cilia (Ac-TUB) in Tsc1 − / − and Tsc2 − / − MEFs subjected to siRNA-mediated knock down followed by culturing in starvation medium (0.5% serum) for 48 h (24 h after transfection) to induce cilia formation. Nuclei were visualized with DAPI staining. Scale bar: 5 µM. f Quantification of ciliary lengths for experiment shown in e . One hundred cilia from three independent experiments were used for quantification; error bars represent SEM. g Quantification of ciliary lengths labeled with Ac-TUB antibody in WT, Tsc1 − / − and Tsc2 − / − MEFs cultured in low 48 h in the presence or absence of rapamycin

    Article Snippet: To induce ciliary formation cells were starved (0.5% FBS) for 48 h, and to measure autophagic flux cells were cultured in the presence and in the absence of rapamycin before lysosomal degradation was inhibited by 20 mM ammonium chloride (NH4 Cl, Sigma-Aldrich #A-0171) and 200 µm Leupeptin (Biovision #1648) for 2 or 4 h. Cells were treated with 25 nM rapamycin (Cell Signaling Technology #9904) for 48 h to inhibit mTORC1 activity and 5 µM purmorphamine (Santa Cruz biotechnologies #sc-202785) for 24 h to activate HH signaling (last 24 h of the 48 h in starvation medium).

    Techniques: Expressing, Marker, Labeling, Cell Culture, Western Blot, Transfection, Negative Control, Staining

    Loss of Tsc1 or Tsc2 is associated with impaired HH signaling. a – d WT, Tsc1 − / − and Tsc2 − / − MEFs were starved (0.5% FBS) for 48 h to induce cilia formation in the presence or absence of rapamycin. Purmorphamine (5 µM) was administered to the medium for the final 24 h to stimulate HH signaling before mRNA isolation and qPCR analysis. Expression profiles of the target genes were normalized to the amount of endogenous Tbp mRNA. Normalized expression profiles of a Gli1 , b Ptch1. Error bars represent SEM ( n = 5). Normalized expression profiles of c Gli2 and d Gli3 . e Normalized expression profiles of Gli1 in Tsc1 − / − MEFs transfected with 2 µg plasmid (pMock (empty vector) or pGli2) and cultured in starvation medium (0.5% FBS) for 48 h (24 h after transfection) with purmorphamine administration (for the last 24 h), as indicated. Error bars represent SEM ( n = 3). The reduced signal in e compared to a might be due to transfection (pMock included) of the cells. f Normalized expression profiles of Gli2 in Tsc1 − / − MEFs transfected with 1 µg plasmid (pMock or pTSC1) and starved (0.5% serum) for 48 h (24 h after transfection). Error bars represent SEM ( n = 3)

    Journal: Cellular and Molecular Life Sciences

    Article Title: TSC1 and TSC2 regulate cilia length and canonical Hedgehog signaling via different mechanisms

    doi: 10.1007/s00018-018-2761-8

    Figure Lengend Snippet: Loss of Tsc1 or Tsc2 is associated with impaired HH signaling. a – d WT, Tsc1 − / − and Tsc2 − / − MEFs were starved (0.5% FBS) for 48 h to induce cilia formation in the presence or absence of rapamycin. Purmorphamine (5 µM) was administered to the medium for the final 24 h to stimulate HH signaling before mRNA isolation and qPCR analysis. Expression profiles of the target genes were normalized to the amount of endogenous Tbp mRNA. Normalized expression profiles of a Gli1 , b Ptch1. Error bars represent SEM ( n = 5). Normalized expression profiles of c Gli2 and d Gli3 . e Normalized expression profiles of Gli1 in Tsc1 − / − MEFs transfected with 2 µg plasmid (pMock (empty vector) or pGli2) and cultured in starvation medium (0.5% FBS) for 48 h (24 h after transfection) with purmorphamine administration (for the last 24 h), as indicated. Error bars represent SEM ( n = 3). The reduced signal in e compared to a might be due to transfection (pMock included) of the cells. f Normalized expression profiles of Gli2 in Tsc1 − / − MEFs transfected with 1 µg plasmid (pMock or pTSC1) and starved (0.5% serum) for 48 h (24 h after transfection). Error bars represent SEM ( n = 3)

    Article Snippet: To induce ciliary formation cells were starved (0.5% FBS) for 48 h, and to measure autophagic flux cells were cultured in the presence and in the absence of rapamycin before lysosomal degradation was inhibited by 20 mM ammonium chloride (NH4 Cl, Sigma-Aldrich #A-0171) and 200 µm Leupeptin (Biovision #1648) for 2 or 4 h. Cells were treated with 25 nM rapamycin (Cell Signaling Technology #9904) for 48 h to inhibit mTORC1 activity and 5 µM purmorphamine (Santa Cruz biotechnologies #sc-202785) for 24 h to activate HH signaling (last 24 h of the 48 h in starvation medium).

    Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, Cell Culture

    Loss of Tsc1 leads to increased mTORC1-dependent autophagic activity. a SDS-PAGE and WB analyses on the level of LC3B and phosphoS6 (pS6) in WT, Tsc1 − / − and Tsc2 − / − MEFs starved (0.5% FBS) for 48 h in the absence or presence of rapamycin (Rapa) (24 h) followed by treatment with lysosomal protease inhibitors (PI) as indicated. α-tubulin (α-TUB) was used as loading control. b Normalized LC3B-II flux for experiments shown in a as measured by the LC3B-II protein levels after 2 h PI treatment subtracted the protein levels at 0 h. c Normalized LC3B-I protein levels for experiments shown in a before PI treatment. d Normalized LC3B-II protein levels for experiments shown in a before PI treatment. b – d Quantifications of LC3B-I/II protein levels were performed by densitometric analysis and normalized to α-TUB, and to a control sample loaded on all gels to correct for blotting efficiency. Error bars represent SEM ( n = 3). Recalculating of the LC3B-II flux relative to the amount of LC3B-I eliminated the difference between the cell lines and the effect of rapamycin on the Tsc1 − / − cells (WT: 0.506 ± 0.087/0.584 ± 0.864; Tsc1 − / − : 0.6261 ± 0.314/0.864 ± 0.443; Tsc2 − / − : 0.673 ± 0.253/0.662 ± 0.314. No statistic significant differences were obtained). e IFM analysis of primary cilia labeled with anti-γ-tubulin (γ-TUB) antibody in WT, Tsc1 − / − and Tsc2 − / − MEFs after 4 h PI treatment. Scale bar: 5 µM. f Quantification of ciliary lengths for experiment shown in e . One hundred cilia from three independent experiments were used for quantification; error bars represent SEM. g IFM analysis of primary cilia labeled with γ-TUB antibody in serum-deprived (48 h) WT and Tsc1 − / − MEFs in the presence and in the absence of the autophagy inducer ABT-737. Scale bar: 5 µM. h Quantification of ciliary lengths for experiment shown in g . One hundred cilia from two independent experiments were used for quantification; error bars represent SEM

    Journal: Cellular and Molecular Life Sciences

    Article Title: TSC1 and TSC2 regulate cilia length and canonical Hedgehog signaling via different mechanisms

    doi: 10.1007/s00018-018-2761-8

    Figure Lengend Snippet: Loss of Tsc1 leads to increased mTORC1-dependent autophagic activity. a SDS-PAGE and WB analyses on the level of LC3B and phosphoS6 (pS6) in WT, Tsc1 − / − and Tsc2 − / − MEFs starved (0.5% FBS) for 48 h in the absence or presence of rapamycin (Rapa) (24 h) followed by treatment with lysosomal protease inhibitors (PI) as indicated. α-tubulin (α-TUB) was used as loading control. b Normalized LC3B-II flux for experiments shown in a as measured by the LC3B-II protein levels after 2 h PI treatment subtracted the protein levels at 0 h. c Normalized LC3B-I protein levels for experiments shown in a before PI treatment. d Normalized LC3B-II protein levels for experiments shown in a before PI treatment. b – d Quantifications of LC3B-I/II protein levels were performed by densitometric analysis and normalized to α-TUB, and to a control sample loaded on all gels to correct for blotting efficiency. Error bars represent SEM ( n = 3). Recalculating of the LC3B-II flux relative to the amount of LC3B-I eliminated the difference between the cell lines and the effect of rapamycin on the Tsc1 − / − cells (WT: 0.506 ± 0.087/0.584 ± 0.864; Tsc1 − / − : 0.6261 ± 0.314/0.864 ± 0.443; Tsc2 − / − : 0.673 ± 0.253/0.662 ± 0.314. No statistic significant differences were obtained). e IFM analysis of primary cilia labeled with anti-γ-tubulin (γ-TUB) antibody in WT, Tsc1 − / − and Tsc2 − / − MEFs after 4 h PI treatment. Scale bar: 5 µM. f Quantification of ciliary lengths for experiment shown in e . One hundred cilia from three independent experiments were used for quantification; error bars represent SEM. g IFM analysis of primary cilia labeled with γ-TUB antibody in serum-deprived (48 h) WT and Tsc1 − / − MEFs in the presence and in the absence of the autophagy inducer ABT-737. Scale bar: 5 µM. h Quantification of ciliary lengths for experiment shown in g . One hundred cilia from two independent experiments were used for quantification; error bars represent SEM

    Article Snippet: To induce ciliary formation cells were starved (0.5% FBS) for 48 h, and to measure autophagic flux cells were cultured in the presence and in the absence of rapamycin before lysosomal degradation was inhibited by 20 mM ammonium chloride (NH4 Cl, Sigma-Aldrich #A-0171) and 200 µm Leupeptin (Biovision #1648) for 2 or 4 h. Cells were treated with 25 nM rapamycin (Cell Signaling Technology #9904) for 48 h to inhibit mTORC1 activity and 5 µM purmorphamine (Santa Cruz biotechnologies #sc-202785) for 24 h to activate HH signaling (last 24 h of the 48 h in starvation medium).

    Techniques: Activity Assay, SDS Page, Western Blot, Labeling

    Effects of rapamycin or chloroquine on autophagic activity and EG-induced CaOx crystals depositions in rat kidneys. (A) Quantitative changes in autophagic vacuoles under TEM in kidney sections from indicated rats. The red arrows indicate autophagic vacuoles. Scale bar: 1 µm. (B) The expressions of Beclin1 and SQSTM1 in kidney sections from indicated rats were detected by immunohistochemical staining and quantified using Image Pro Plus software. (magnification 200×) (C) Photomicrographs of kidney sections from indicated rats were obtained under dark field illumination with polarized light. Retained crystals exhibit strong birefringence (magnification 200×). The sizes of the areas of crystal deposition per field were estimated and quantified using ImageJ software. * P

    Journal: Redox Biology

    Article Title: Autophagy inhibition attenuates hyperoxaluria-induced renal tubular oxidative injury and calcium oxalate crystal depositions in the rat kidney

    doi: 10.1016/j.redox.2018.03.019

    Figure Lengend Snippet: Effects of rapamycin or chloroquine on autophagic activity and EG-induced CaOx crystals depositions in rat kidneys. (A) Quantitative changes in autophagic vacuoles under TEM in kidney sections from indicated rats. The red arrows indicate autophagic vacuoles. Scale bar: 1 µm. (B) The expressions of Beclin1 and SQSTM1 in kidney sections from indicated rats were detected by immunohistochemical staining and quantified using Image Pro Plus software. (magnification 200×) (C) Photomicrographs of kidney sections from indicated rats were obtained under dark field illumination with polarized light. Retained crystals exhibit strong birefringence (magnification 200×). The sizes of the areas of crystal deposition per field were estimated and quantified using ImageJ software. * P

    Article Snippet: The rats in the treatment groups were daily intraperitoneally injected with chloroquine (30 mg/kg/d, Sigma-Aldrich, USA) or rapamycin (0.25 mg/kg/d, Sigma-Aldrich, USA) for 4 weeks, whereas the control rats received an equal volume of normal saline.

    Techniques: Activity Assay, Transmission Electron Microscopy, Immunohistochemistry, Staining, Software

    Effects of rapamycin or chloroquine on EG-induced renal oxidative injury in rat kidneys. (A) Immunohistochemical staining and quantification for oxidative injury-related markers and p38 in kidney sections from indicated rats. Original magnification 200×. (B) Ultrastructural images of mitochondria in kidney sections from indicated rats. The red arrows indicate mitochondria. Scale bar: 500 nm. The surface areas of indicated mitochondria were quantified using ImageJ software. * P

    Journal: Redox Biology

    Article Title: Autophagy inhibition attenuates hyperoxaluria-induced renal tubular oxidative injury and calcium oxalate crystal depositions in the rat kidney

    doi: 10.1016/j.redox.2018.03.019

    Figure Lengend Snippet: Effects of rapamycin or chloroquine on EG-induced renal oxidative injury in rat kidneys. (A) Immunohistochemical staining and quantification for oxidative injury-related markers and p38 in kidney sections from indicated rats. Original magnification 200×. (B) Ultrastructural images of mitochondria in kidney sections from indicated rats. The red arrows indicate mitochondria. Scale bar: 500 nm. The surface areas of indicated mitochondria were quantified using ImageJ software. * P

    Article Snippet: The rats in the treatment groups were daily intraperitoneally injected with chloroquine (30 mg/kg/d, Sigma-Aldrich, USA) or rapamycin (0.25 mg/kg/d, Sigma-Aldrich, USA) for 4 weeks, whereas the control rats received an equal volume of normal saline.

    Techniques: Immunohistochemistry, Staining, Software

    Effects of rapamycin and chloroquine on oxalate-induced oxidative injury of NRK-52E cells. (A-C) Twenty-four hours after being seeded in a 96-well plate, NRK-52Ecells were incubated with or without rapamycin (RAP, 5 μM) or chloroquine (Clo, 5 μM) for 2 h, after which they were stimulated with oxalate (0.75 mM) for 48 h. Cell viability, LDH release and GSH release were subsequently assessed as described in the “Materials and Methods”. (D) DHE staining results and quantitative data for cells treated as described in (A-C). Original magnification 200×. (E) Ultrastructural images of the mitochondria in NRK-52E cells treated as described above. Scale bar: 500 nm. The surface areas of indicated mitochondria were quantified using ImageJ software. (F) Mitochondrial membrane potential (Δψm) of the cells treated as described above was determined by JC-10 assay. (G) Crystal-cell adhesion assay results and quantitative data for ponceau-S-labeled COM crystals (red) adhering to cells treated as described above. Original magnification 200×. * P

    Journal: Redox Biology

    Article Title: Autophagy inhibition attenuates hyperoxaluria-induced renal tubular oxidative injury and calcium oxalate crystal depositions in the rat kidney

    doi: 10.1016/j.redox.2018.03.019

    Figure Lengend Snippet: Effects of rapamycin and chloroquine on oxalate-induced oxidative injury of NRK-52E cells. (A-C) Twenty-four hours after being seeded in a 96-well plate, NRK-52Ecells were incubated with or without rapamycin (RAP, 5 μM) or chloroquine (Clo, 5 μM) for 2 h, after which they were stimulated with oxalate (0.75 mM) for 48 h. Cell viability, LDH release and GSH release were subsequently assessed as described in the “Materials and Methods”. (D) DHE staining results and quantitative data for cells treated as described in (A-C). Original magnification 200×. (E) Ultrastructural images of the mitochondria in NRK-52E cells treated as described above. Scale bar: 500 nm. The surface areas of indicated mitochondria were quantified using ImageJ software. (F) Mitochondrial membrane potential (Δψm) of the cells treated as described above was determined by JC-10 assay. (G) Crystal-cell adhesion assay results and quantitative data for ponceau-S-labeled COM crystals (red) adhering to cells treated as described above. Original magnification 200×. * P

    Article Snippet: The rats in the treatment groups were daily intraperitoneally injected with chloroquine (30 mg/kg/d, Sigma-Aldrich, USA) or rapamycin (0.25 mg/kg/d, Sigma-Aldrich, USA) for 4 weeks, whereas the control rats received an equal volume of normal saline.

    Techniques: Incubation, Staining, Software, Cell Adhesion Assay, Labeling

    LAM cell medium is chemotactic for fibroblasts, and this is partially dependent on CXCR4. (a) 621–101 cells secrete CXCL12 protein; this is partially sensitive to rapamycin. (b) The presence of 621–101 cell conditioned medium (CM) increases fibroblast migration, which is partially blocked by AMD3100. (c) Fibroblasts migrate towards CXCL12 in a modified Boyden chamber (Transwell) assay; this is inhibited by the CXCR4 receptor antagonist AMD3100 at 100μg/ml (*p = 0.05). (d) Fibroblasts migrate towards 621–101 cells in a bidirectional migration assay. (i) Removal of the fence leaves a gap between the peripheral fibroblasts (green) and the central reservoir of (red) 621–101 cells (dotted), (ii) Fibroblasts migrate towards the central reservoir (arrowed), (iii) fibroblasts migrate into the central area (arrowed). (e) Addition of 200μg/ml AMD3100 reduces migration of fibroblasts in response to 621–101 cells by 56%. In the absence of 621–101 cells in the central reservoir migration is reduced by 93%.

    Journal: PLoS ONE

    Article Title: Wild Type Mesenchymal Cells Contribute to the Lung Pathology of Lymphangioleiomyomatosis

    doi: 10.1371/journal.pone.0126025

    Figure Lengend Snippet: LAM cell medium is chemotactic for fibroblasts, and this is partially dependent on CXCR4. (a) 621–101 cells secrete CXCL12 protein; this is partially sensitive to rapamycin. (b) The presence of 621–101 cell conditioned medium (CM) increases fibroblast migration, which is partially blocked by AMD3100. (c) Fibroblasts migrate towards CXCL12 in a modified Boyden chamber (Transwell) assay; this is inhibited by the CXCR4 receptor antagonist AMD3100 at 100μg/ml (*p = 0.05). (d) Fibroblasts migrate towards 621–101 cells in a bidirectional migration assay. (i) Removal of the fence leaves a gap between the peripheral fibroblasts (green) and the central reservoir of (red) 621–101 cells (dotted), (ii) Fibroblasts migrate towards the central reservoir (arrowed), (iii) fibroblasts migrate into the central area (arrowed). (e) Addition of 200μg/ml AMD3100 reduces migration of fibroblasts in response to 621–101 cells by 56%. In the absence of 621–101 cells in the central reservoir migration is reduced by 93%.

    Article Snippet: For rapamycin inhibition of S6 phosphorylation, medium was again replaced with fresh serum-free DME-F12 then 10nM rapamycin (InSolution Rapamycin, Merck Millipore, Watford, UK) was added 30 minutes prior to 10% foetal bovine serum.

    Techniques: Laser Capture Microdissection, Migration, Modification, Boyden Chamber Transwell Assay

    Rapamycin improves recovery of neurobehavioral function after intracerebral hemorrhage (ICH). The scores in both the ICH and rapamycin groups were similar before ICH. At 1 hour after ICH, rapamycin-treated groups were injected with rapamycin using different concentrations: 50, 150, 250, and 500 μg/kg. The behaviors were evaluated and compared at 1, 3, 7, and 14 days after ICH. We observed a significant functional recovery in rapamycin-treated groups compared with the ICH group. The mean modified Neurological Severity Score (mNSS) values ± SEM are depicted, * P

    Journal: Journal of Neuroinflammation

    Article Title: Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

    doi: 10.1186/1742-2094-11-44

    Figure Lengend Snippet: Rapamycin improves recovery of neurobehavioral function after intracerebral hemorrhage (ICH). The scores in both the ICH and rapamycin groups were similar before ICH. At 1 hour after ICH, rapamycin-treated groups were injected with rapamycin using different concentrations: 50, 150, 250, and 500 μg/kg. The behaviors were evaluated and compared at 1, 3, 7, and 14 days after ICH. We observed a significant functional recovery in rapamycin-treated groups compared with the ICH group. The mean modified Neurological Severity Score (mNSS) values ± SEM are depicted, * P

    Article Snippet: Rapamycin Rapamycin (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO (5 mg/ml) and stored at -20°C.

    Techniques: Injection, Functional Assay, Modification

    Rapamycin increased the level of regulatory T cells (Tregs) in the ipsilateral hemisphere. Dot plots labeled with CD4 and Foxp3 show the brain lymphocytes from (A) control group, (B) intracerebral hemorrhage (ICH) group, and (C) 150 μg/kg rapamycin-treated group. (D) A statistical graph for the three groups. There were significantly higher levels of Tregs in the rapamycin-treated group than the control and ICH groups. * P

    Journal: Journal of Neuroinflammation

    Article Title: Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

    doi: 10.1186/1742-2094-11-44

    Figure Lengend Snippet: Rapamycin increased the level of regulatory T cells (Tregs) in the ipsilateral hemisphere. Dot plots labeled with CD4 and Foxp3 show the brain lymphocytes from (A) control group, (B) intracerebral hemorrhage (ICH) group, and (C) 150 μg/kg rapamycin-treated group. (D) A statistical graph for the three groups. There were significantly higher levels of Tregs in the rapamycin-treated group than the control and ICH groups. * P

    Article Snippet: Rapamycin Rapamycin (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO (5 mg/ml) and stored at -20°C.

    Techniques: Labeling

    Effect of rapamycin on the levels of interferon (IFN)-γ and interleukin (IL)-10 at an autologous blood-injection model of intracerebral hemorrhage (ICH). (A, B) Similar to the collagenase-injection model, the levels of IFN-γ was downregulated after treatment with rapamycin both in serum and around the hematoma in an autologous blood-injection model. (C, D) Rapamycin upregulated IL-10 both in serum and around the hematoma. AU, Autologous blood-injection model of ICH, CO, Collagenase-injection model of ICH, RA, Rapamycin. * P

    Journal: Journal of Neuroinflammation

    Article Title: Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

    doi: 10.1186/1742-2094-11-44

    Figure Lengend Snippet: Effect of rapamycin on the levels of interferon (IFN)-γ and interleukin (IL)-10 at an autologous blood-injection model of intracerebral hemorrhage (ICH). (A, B) Similar to the collagenase-injection model, the levels of IFN-γ was downregulated after treatment with rapamycin both in serum and around the hematoma in an autologous blood-injection model. (C, D) Rapamycin upregulated IL-10 both in serum and around the hematoma. AU, Autologous blood-injection model of ICH, CO, Collagenase-injection model of ICH, RA, Rapamycin. * P

    Article Snippet: Rapamycin Rapamycin (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO (5 mg/ml) and stored at -20°C.

    Techniques: Injection

    Rapamycin increased the level of regulatory T cells (Tregs) in the blood. Dot plots labeled with CD4 and Foxp3 show the blood lymphocytes derived from (A) control group, (B) intracerebral hemorrhage (ICH) group, and (C) 150 μg/kg rapamycin-treated group. (D) A statistical graph for the three groups. There were significant differences between the control group and rapamycin-treated groups, and between the ICH and rapamycin-treated groups. *P

    Journal: Journal of Neuroinflammation

    Article Title: Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

    doi: 10.1186/1742-2094-11-44

    Figure Lengend Snippet: Rapamycin increased the level of regulatory T cells (Tregs) in the blood. Dot plots labeled with CD4 and Foxp3 show the blood lymphocytes derived from (A) control group, (B) intracerebral hemorrhage (ICH) group, and (C) 150 μg/kg rapamycin-treated group. (D) A statistical graph for the three groups. There were significant differences between the control group and rapamycin-treated groups, and between the ICH and rapamycin-treated groups. *P

    Article Snippet: Rapamycin Rapamycin (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO (5 mg/ml) and stored at -20°C.

    Techniques: Labeling, Derivative Assay

    Levels of cytokines in the serum after rapamycin treatment. (A) The levels of interferon (IFN)-γ in each rapamycin-treated group were lower than the intracerebral hemorrhage (ICH) group. (B) Rapamycin-treated groups presented higher interleukin (IL)-10 expression than the ICH group. (C) The ratio of interleukin (IL)-10 to IFN-γ increased after treatment with rapamycin (150, 250, and 500 μg/kg, but not 50 μg/kg). (D) Rapamycin increased the level of transforming growth factor (TGF)-β. There were no significant differences between the 150, 250, and 500 μg/kg rapamycin-treated groups. * P

    Journal: Journal of Neuroinflammation

    Article Title: Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

    doi: 10.1186/1742-2094-11-44

    Figure Lengend Snippet: Levels of cytokines in the serum after rapamycin treatment. (A) The levels of interferon (IFN)-γ in each rapamycin-treated group were lower than the intracerebral hemorrhage (ICH) group. (B) Rapamycin-treated groups presented higher interleukin (IL)-10 expression than the ICH group. (C) The ratio of interleukin (IL)-10 to IFN-γ increased after treatment with rapamycin (150, 250, and 500 μg/kg, but not 50 μg/kg). (D) Rapamycin increased the level of transforming growth factor (TGF)-β. There were no significant differences between the 150, 250, and 500 μg/kg rapamycin-treated groups. * P

    Article Snippet: Rapamycin Rapamycin (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO (5 mg/ml) and stored at -20°C.

    Techniques: Expressing

    Increased mammalian target of rapamycin (mTOR) activation in the ipsilateral striatum after intracerebral hemorrhage (ICH). (A) Protein from the striatum was analyzed by western blotting using anti-p-mTOR (Ser 2448). The level of p-mTOR was normalized to the level of tubulin. The p-mTOR significantly increased at 30 minutes after ICH, and returned to basal level at 1 day after ICH. (B) The change in total mTOR was not significant after being normalized to tubulin. * P

    Journal: Journal of Neuroinflammation

    Article Title: Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

    doi: 10.1186/1742-2094-11-44

    Figure Lengend Snippet: Increased mammalian target of rapamycin (mTOR) activation in the ipsilateral striatum after intracerebral hemorrhage (ICH). (A) Protein from the striatum was analyzed by western blotting using anti-p-mTOR (Ser 2448). The level of p-mTOR was normalized to the level of tubulin. The p-mTOR significantly increased at 30 minutes after ICH, and returned to basal level at 1 day after ICH. (B) The change in total mTOR was not significant after being normalized to tubulin. * P

    Article Snippet: Rapamycin Rapamycin (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO (5 mg/ml) and stored at -20°C.

    Techniques: Activation Assay, Western Blot

    Mammalian target of rapamycin (mTOR) activation in the ipsilateral cortex after intracerebral hemorrhage (ICH). The levels of both (A) p-mTOR and (B) total mTOR did not show significant changes after ICH (after normalization to tubulin).

    Journal: Journal of Neuroinflammation

    Article Title: Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

    doi: 10.1186/1742-2094-11-44

    Figure Lengend Snippet: Mammalian target of rapamycin (mTOR) activation in the ipsilateral cortex after intracerebral hemorrhage (ICH). The levels of both (A) p-mTOR and (B) total mTOR did not show significant changes after ICH (after normalization to tubulin).

    Article Snippet: Rapamycin Rapamycin (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO (5 mg/ml) and stored at -20°C.

    Techniques: Activation Assay

    Rapamycin inhibited p-mammalian target of rapamycin (mTOR) in the striatum. (A) Compared with the intracerebral hemorrhage (ICH) group (4 h after ICH), significantly lower levels of p-mTOR were observed in the 150, 250, and 500 μg/kg treated groups while there was no significant change in the 50 μg/kg treated group. (B) The total mTOR level in rapamycin-treated groups was similar to that of the ICH group. Both p-mTOR and total mTOR were normalized to tubulin. * P

    Journal: Journal of Neuroinflammation

    Article Title: Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

    doi: 10.1186/1742-2094-11-44

    Figure Lengend Snippet: Rapamycin inhibited p-mammalian target of rapamycin (mTOR) in the striatum. (A) Compared with the intracerebral hemorrhage (ICH) group (4 h after ICH), significantly lower levels of p-mTOR were observed in the 150, 250, and 500 μg/kg treated groups while there was no significant change in the 50 μg/kg treated group. (B) The total mTOR level in rapamycin-treated groups was similar to that of the ICH group. Both p-mTOR and total mTOR were normalized to tubulin. * P

    Article Snippet: Rapamycin Rapamycin (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO (5 mg/ml) and stored at -20°C.

    Techniques:

    Mammalian target of rapamycin (mTOR) was activated after intracerebral hemorrhage (ICH). (A) Protein from the ipsilateral hemisphere was analyzed by western blotting using anti-p-mTOR (Ser 2448)., which was was greatly increased at 30 minutes after ICH and lasted up to 14 days after ICH. The level of p-mTOR was normalized to the level of tubulin. * P

    Journal: Journal of Neuroinflammation

    Article Title: Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

    doi: 10.1186/1742-2094-11-44

    Figure Lengend Snippet: Mammalian target of rapamycin (mTOR) was activated after intracerebral hemorrhage (ICH). (A) Protein from the ipsilateral hemisphere was analyzed by western blotting using anti-p-mTOR (Ser 2448)., which was was greatly increased at 30 minutes after ICH and lasted up to 14 days after ICH. The level of p-mTOR was normalized to the level of tubulin. * P

    Article Snippet: Rapamycin Rapamycin (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO (5 mg/ml) and stored at -20°C.

    Techniques: Western Blot

    Levels of cytokines around the hematoma after rapamycin treatment. (A) Compared with the intracerebral hemorrhage (ICH) group, the levels of interferon (IFN)- γ were reduced in the rapamycin-treated groups except for the 50 μg/kg group. (B) Rapamycin-treated groups had higher levels of interleukin ( IL ) -10 than the ICH group, except for the 50 μg/kg group. (C) The ratio of IL-10 to IFN-γ was increased after treatment with rapamycin (150, 250, and 500 μg/kg, but not 50 μg/kg). (D) Rapamycin increased the level of transforming growth factor (TGF)-β. * P

    Journal: Journal of Neuroinflammation

    Article Title: Inhibition of mammalian target of rapamycin improves neurobehavioral deficit and modulates immune response after intracerebral hemorrhage in rat

    doi: 10.1186/1742-2094-11-44

    Figure Lengend Snippet: Levels of cytokines around the hematoma after rapamycin treatment. (A) Compared with the intracerebral hemorrhage (ICH) group, the levels of interferon (IFN)- γ were reduced in the rapamycin-treated groups except for the 50 μg/kg group. (B) Rapamycin-treated groups had higher levels of interleukin ( IL ) -10 than the ICH group, except for the 50 μg/kg group. (C) The ratio of IL-10 to IFN-γ was increased after treatment with rapamycin (150, 250, and 500 μg/kg, but not 50 μg/kg). (D) Rapamycin increased the level of transforming growth factor (TGF)-β. * P

    Article Snippet: Rapamycin Rapamycin (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO (5 mg/ml) and stored at -20°C.

    Techniques:

    Targeting FAK via RNAi enhanced the growth-inhibitory effects of rapamycin. a FAK protein ( i ) and mRNA expression ( ii ) in REH-empty vector and REH-FAK shRNA cells. b The mTOR inhibitor rapamycin and FAK down-regulation inhibited the proliferation of REH cells. REH-empty vector or REH-FAK shRNA cells were plated in 96-well plates and cultured with rapamycin (0–1000 nM). After 2 days ( i ) or 3 days ( ii ), the cells were treated with CCK-8 (10 μl/well) for 2 h and the absorbance was measured. The results represent the mean ± S.D. of three experiments performed in triplicate

    Journal: Journal of Hematology & Oncology

    Article Title: Synergism between the mTOR inhibitor rapamycin and FAK down-regulation in the treatment of acute lymphoblastic leukemia

    doi: 10.1186/s13045-016-0241-x

    Figure Lengend Snippet: Targeting FAK via RNAi enhanced the growth-inhibitory effects of rapamycin. a FAK protein ( i ) and mRNA expression ( ii ) in REH-empty vector and REH-FAK shRNA cells. b The mTOR inhibitor rapamycin and FAK down-regulation inhibited the proliferation of REH cells. REH-empty vector or REH-FAK shRNA cells were plated in 96-well plates and cultured with rapamycin (0–1000 nM). After 2 days ( i ) or 3 days ( ii ), the cells were treated with CCK-8 (10 μl/well) for 2 h and the absorbance was measured. The results represent the mean ± S.D. of three experiments performed in triplicate

    Article Snippet: Reagents Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, was purchased from Sigma (USA).

    Techniques: Expressing, Plasmid Preparation, shRNA, Cell Culture, CCK-8 Assay

    Low doses of Rapamycin, Torin 1, and Torin 2 inhibit mTOR and induce autophagy. (A) RAW264.7 cells were pretreated with 1 uM or 10 uM of the mTOR inhibitors indicated and then challenged with 1 ug/ml E. coli derived LPS for 3 hours. Protein lysates were prepared and western blots for total ribosomal S6 and phosphorylated ribosomal S6 are shown. Shown are data representative of two independent assays (B) RAW264.7 cells were infected with M. smegmatis (MOI 5) and treated with the mTOR inhibitors shown. Protein lysates were prepared and western blots for Actin and phosphorylated ribosomal S6 were performed. Shown are data representative of two independent assays (C) A549 cells were treated with 10 uM of the indicated inhibitor for 3 hours and then stained for endogenous LC3B, or an isotype control IgG, and imaged by fluorescence microscopy. Shown are data representative of two independent assays. (D) RAW264.7 cells were loaded with DQ-BSA, either left untreated (−DMSO) or treated overnight with the indicated concentrations of the mTOR inhibitors shown, and analyzed by flow cytometry. Shown is the combined percentage of DQ-BSA positive cells (+/− SEM) and the mean fluorescent intensity (and intensity range) derived from two independent assays with 3 replicates per assay. For analysis of the percent DQ-BSA positive cells, asterisks indicated p

    Journal: BMC Biochemistry

    Article Title: Mammalian target of Rapamycin inhibition and mycobacterial survival are uncoupled in murine macrophages

    doi: 10.1186/1471-2091-15-4

    Figure Lengend Snippet: Low doses of Rapamycin, Torin 1, and Torin 2 inhibit mTOR and induce autophagy. (A) RAW264.7 cells were pretreated with 1 uM or 10 uM of the mTOR inhibitors indicated and then challenged with 1 ug/ml E. coli derived LPS for 3 hours. Protein lysates were prepared and western blots for total ribosomal S6 and phosphorylated ribosomal S6 are shown. Shown are data representative of two independent assays (B) RAW264.7 cells were infected with M. smegmatis (MOI 5) and treated with the mTOR inhibitors shown. Protein lysates were prepared and western blots for Actin and phosphorylated ribosomal S6 were performed. Shown are data representative of two independent assays (C) A549 cells were treated with 10 uM of the indicated inhibitor for 3 hours and then stained for endogenous LC3B, or an isotype control IgG, and imaged by fluorescence microscopy. Shown are data representative of two independent assays. (D) RAW264.7 cells were loaded with DQ-BSA, either left untreated (−DMSO) or treated overnight with the indicated concentrations of the mTOR inhibitors shown, and analyzed by flow cytometry. Shown is the combined percentage of DQ-BSA positive cells (+/− SEM) and the mean fluorescent intensity (and intensity range) derived from two independent assays with 3 replicates per assay. For analysis of the percent DQ-BSA positive cells, asterisks indicated p

    Article Snippet: mTOR inhibitors Rapamycin (Sigma), Torin 1, and Torin 2, (Tocris) were dissolved in DMSO to a concentration of 10 mM, aliquoted, and stored at −20°C.

    Techniques: Derivative Assay, Western Blot, Infection, Staining, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    IL-2 activates STAT3 pathway to induce the in vitro maturation of hIOs. a ELISA quantification of IL-2, IL-8, TNFα, IL-22, IL-6, IL-1β, IL-11, EGF, OSM, and IL-10 concentrations in the culture supernatant of stimulated and unstimulated Jurkat T cells. Expression of IL-2R subunits as analyzed by RT-PCR b and Western blot analyses c in co-cultured or IL-2-treated hIOs. d Phosphorylation levels of proteins from multiple signaling pathways in control, co-cultured and IL-2-treated hIOs as reported by a human phospho-kinase array (upper panels). Analysis by functional interaction (FI) network highlighted a significant enrichment of phospho-proteins in the mTOR and STAT3 signaling pathways (lower panels). In the FI network, arrows represent activating/catalyzing connections, solid lines ending in a perpendicular line represent inhibition, solid lines represent complexes or inputs and dashed lines represent predicted FI connections. e Representative images of the morphology of hIOs cultured in the presence of 1 ng/ml IL-2, a key component of the co-culture system, or stimulated Jurkat T conditioned medium (CM) with or without IL-2R-inactivating antibodies (anti-IL-2Rβ, anti-IL-2Rγ c ) for two passages. Quantitative assessment of the size of hIOs (left bottom) and the number of budding structure per hIO (right bottom); n = 12 hIOs per group. f Representative Western blot analysis of p-STAT3, p-AKT and p-P70 S6 Kinase levels in co-cultured and IL-2-treated hIOs. g hIOs cultured in the presence of IL-2 (1 ng/ml) with or without the addition of S3I-201 (5 μM), Stattic (1 μM) or Rapamycin (Rapa; 10 nM). Quantitative assessment of the size of hIOs after one passage (14 days) (left bottom) and the number of budding structure per hIO (right bottom); n = 14 hIOs per group. Data are presented as mean values of replicates ± SEM. *** p

    Journal: Nature Communications

    Article Title: Interleukin-2 induces the in vitro maturation of human pluripotent stem cell-derived intestinal organoids

    doi: 10.1038/s41467-018-05450-8

    Figure Lengend Snippet: IL-2 activates STAT3 pathway to induce the in vitro maturation of hIOs. a ELISA quantification of IL-2, IL-8, TNFα, IL-22, IL-6, IL-1β, IL-11, EGF, OSM, and IL-10 concentrations in the culture supernatant of stimulated and unstimulated Jurkat T cells. Expression of IL-2R subunits as analyzed by RT-PCR b and Western blot analyses c in co-cultured or IL-2-treated hIOs. d Phosphorylation levels of proteins from multiple signaling pathways in control, co-cultured and IL-2-treated hIOs as reported by a human phospho-kinase array (upper panels). Analysis by functional interaction (FI) network highlighted a significant enrichment of phospho-proteins in the mTOR and STAT3 signaling pathways (lower panels). In the FI network, arrows represent activating/catalyzing connections, solid lines ending in a perpendicular line represent inhibition, solid lines represent complexes or inputs and dashed lines represent predicted FI connections. e Representative images of the morphology of hIOs cultured in the presence of 1 ng/ml IL-2, a key component of the co-culture system, or stimulated Jurkat T conditioned medium (CM) with or without IL-2R-inactivating antibodies (anti-IL-2Rβ, anti-IL-2Rγ c ) for two passages. Quantitative assessment of the size of hIOs (left bottom) and the number of budding structure per hIO (right bottom); n = 12 hIOs per group. f Representative Western blot analysis of p-STAT3, p-AKT and p-P70 S6 Kinase levels in co-cultured and IL-2-treated hIOs. g hIOs cultured in the presence of IL-2 (1 ng/ml) with or without the addition of S3I-201 (5 μM), Stattic (1 μM) or Rapamycin (Rapa; 10 nM). Quantitative assessment of the size of hIOs after one passage (14 days) (left bottom) and the number of budding structure per hIO (right bottom); n = 14 hIOs per group. Data are presented as mean values of replicates ± SEM. *** p

    Article Snippet: To block IL-2 downstream signal transduction, either the mTOR inhibitor Rapamycin (10 nM; Sigma-Aldrich) or one of the STAT3 inhibitors S3I-201 (10 μM; Sigma-Aldrich) or Stattic (1 μM; Sigma-Aldrich) were also added.

    Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Functional Assay, Inhibition, Co-Culture Assay

    PMT stimulated release of TNF-α or IL-6 is independent of the mTOR pathway. RAW264.7 cells were stimulated with PMT (5 nM) w or w/o pre-incubation with rapamycin (10 ng/ml) for 1 h. As control, cells were untreated, treated with the solvent control DMSO, with rapamycin or LPS (100 ng/ml) as positive control. After 24 h, supernatants were removed and subjected to ELISA for the measurement of ( a ) IL-6-, ( b ) TNF-α-, ( c ) IL12p40- or ( d ) IL-10 release. Shown are the results of three independent experiments (mean ± SD; n = 3)

    Journal: Cell Communication and Signaling : CCS

    Article Title: Pasteurella multocida toxin- induced osteoclastogenesis requires mTOR activation

    doi: 10.1186/s12964-015-0117-7

    Figure Lengend Snippet: PMT stimulated release of TNF-α or IL-6 is independent of the mTOR pathway. RAW264.7 cells were stimulated with PMT (5 nM) w or w/o pre-incubation with rapamycin (10 ng/ml) for 1 h. As control, cells were untreated, treated with the solvent control DMSO, with rapamycin or LPS (100 ng/ml) as positive control. After 24 h, supernatants were removed and subjected to ELISA for the measurement of ( a ) IL-6-, ( b ) TNF-α-, ( c ) IL12p40- or ( d ) IL-10 release. Shown are the results of three independent experiments (mean ± SD; n = 3)

    Article Snippet: The mTOR Inhibitor I (rapamycin) was purchased from Calbiochem and used at 10 ng/ml.

    Techniques: Incubation, Positive Control, Enzyme-linked Immunosorbent Assay

    Osteoclast differentiation of RAW264.7 cells with PMT requires mTOR activation and shows a distinct morphology. ( a ) RAW264.7 cells were stimulated with PMT (5 nM) or RANKL (50 ng/ml) as a positive control for osteoclast differentiation. After 4 days of stimulation, cells were labelled in an enzymatic reaction for the activity of the enzyme TRAP (ELF97) in green. Nuclei were stained with DAPI (blue) and the cytoskeleton with TRITC-Phalloidin (red). Pictures were taken by confocal laser scanning microscopy and representative pictures are shown (number of independent experiments n ≥3). ( b ) RAW264.7 cells were seeded in 6-well plates, stimulated with 5 nM PMT or 50 ng/ml RANKL for 24 h-48 h before preparation of cDNAs. Quantitative RT-PCR was performed and the graphs display the relative expression for Oscar , Nfatc1 , Ctsk (cathepsin K), Acp5 (TRAP) and ATP6v0d2 normalized to S29 expression. The indicated standard deviation was obtained from four experiments (mean ± SD; n = 4 and n = 3 for Nfatc1 ). Statistical analysis was performed using an unpaired Student’s t -test comparing gene expression to the untreated sample (*: p ≤ 0.05; **: p ≤ 0.005; ***: p ≤ 0.0005). ( c ) The numbers of conventionally TRAP-stained cells were quantified after 3 days of stimulation with the indicated concentrations of PMT or 50 ng/ml RANKL in the presence or absence of rapamycin (10 ng/ml). The graph displays the number of TRAP-positive, multinucleated (≥3) cells. The indicated standard deviation was calculated from three independent experiments (mean ± SD; n = 3). Statistical analysis was performed using an unpaired Student’s t -test (***: p ≤ 0.0005)

    Journal: Cell Communication and Signaling : CCS

    Article Title: Pasteurella multocida toxin- induced osteoclastogenesis requires mTOR activation

    doi: 10.1186/s12964-015-0117-7

    Figure Lengend Snippet: Osteoclast differentiation of RAW264.7 cells with PMT requires mTOR activation and shows a distinct morphology. ( a ) RAW264.7 cells were stimulated with PMT (5 nM) or RANKL (50 ng/ml) as a positive control for osteoclast differentiation. After 4 days of stimulation, cells were labelled in an enzymatic reaction for the activity of the enzyme TRAP (ELF97) in green. Nuclei were stained with DAPI (blue) and the cytoskeleton with TRITC-Phalloidin (red). Pictures were taken by confocal laser scanning microscopy and representative pictures are shown (number of independent experiments n ≥3). ( b ) RAW264.7 cells were seeded in 6-well plates, stimulated with 5 nM PMT or 50 ng/ml RANKL for 24 h-48 h before preparation of cDNAs. Quantitative RT-PCR was performed and the graphs display the relative expression for Oscar , Nfatc1 , Ctsk (cathepsin K), Acp5 (TRAP) and ATP6v0d2 normalized to S29 expression. The indicated standard deviation was obtained from four experiments (mean ± SD; n = 4 and n = 3 for Nfatc1 ). Statistical analysis was performed using an unpaired Student’s t -test comparing gene expression to the untreated sample (*: p ≤ 0.05; **: p ≤ 0.005; ***: p ≤ 0.0005). ( c ) The numbers of conventionally TRAP-stained cells were quantified after 3 days of stimulation with the indicated concentrations of PMT or 50 ng/ml RANKL in the presence or absence of rapamycin (10 ng/ml). The graph displays the number of TRAP-positive, multinucleated (≥3) cells. The indicated standard deviation was calculated from three independent experiments (mean ± SD; n = 3). Statistical analysis was performed using an unpaired Student’s t -test (***: p ≤ 0.0005)

    Article Snippet: The mTOR Inhibitor I (rapamycin) was purchased from Calbiochem and used at 10 ng/ml.

    Techniques: Activation Assay, Positive Control, Activity Assay, Staining, Confocal Laser Scanning Microscopy, Quantitative RT-PCR, Expressing, Standard Deviation

    The mTOR pathway is activated in response to PMT. RAW264.7 cells were stimulated with PMT (5 nM) w or w/o rapamycin (10 ng/ml) for the indicated period of time or were left unstimulated, treated with the solvent control DMSO or rapamycin only. Cells were lysed and the proteins immunoblotted. ( a ) mTOR Ser2448 phosphorylation was determined using a specific antibody and β-actin was used as loading control. ( b ) To investigate the uptake and intracellular action of PMT, cells were stimulated for the indicated time-points with 5 nM PMT and lysed. As a control, cells were stimulated with a catalytically inactive mutant of PMT (PMT C1165S ). The immunoblot was probed with a specific antibody detecting the deamidated form of Gα q (Q209E) or total Gα q . ( c , d ) mTOR-dependent protein phosphorylation from RAW264.7 cells stimulated with PMT was detected using specific antibodies for PRAS40 (Thr246) or p70 S6K1 (Thr389); ß-actin was used as a loading control. The results shown are one representative example of three independent experiments

    Journal: Cell Communication and Signaling : CCS

    Article Title: Pasteurella multocida toxin- induced osteoclastogenesis requires mTOR activation

    doi: 10.1186/s12964-015-0117-7

    Figure Lengend Snippet: The mTOR pathway is activated in response to PMT. RAW264.7 cells were stimulated with PMT (5 nM) w or w/o rapamycin (10 ng/ml) for the indicated period of time or were left unstimulated, treated with the solvent control DMSO or rapamycin only. Cells were lysed and the proteins immunoblotted. ( a ) mTOR Ser2448 phosphorylation was determined using a specific antibody and β-actin was used as loading control. ( b ) To investigate the uptake and intracellular action of PMT, cells were stimulated for the indicated time-points with 5 nM PMT and lysed. As a control, cells were stimulated with a catalytically inactive mutant of PMT (PMT C1165S ). The immunoblot was probed with a specific antibody detecting the deamidated form of Gα q (Q209E) or total Gα q . ( c , d ) mTOR-dependent protein phosphorylation from RAW264.7 cells stimulated with PMT was detected using specific antibodies for PRAS40 (Thr246) or p70 S6K1 (Thr389); ß-actin was used as a loading control. The results shown are one representative example of three independent experiments

    Article Snippet: The mTOR Inhibitor I (rapamycin) was purchased from Calbiochem and used at 10 ng/ml.

    Techniques: Mutagenesis

    PMT-stimulated mTOR activation is required for the full functional activity of the osteoclasts. ( a ) Lysates of cells stimulated for 4 days with PMT concentrations ranging from 10 pM to 5 nM were analyzed for activity of cathepsin K (cleavage of its specific substrate) by fluorescence detection. ( b ) RAW264.7 cells stimulated with PMT (5 nM) w or w/o rapamycin (10 ng/ml) were analyzed for cathepsin K activity by fluorescence detection. As controls, cells were treated with rapamycin, the solvent control DMSO or were left untreated. ( c ) Cells were plated and stimulated on fluoresceinated calcium phosphate plates for 6 days to allow osteoclast differentiation. A fluorescent dye released into the supernatant dependent on the degree of bone resorption was measured with a fluorometer. ( d ) Osteoclasts were differentiated with RANKL and PMT, respectively, on bone slices and the pit area of resorbed bone was determined. ( e ) Bone resorption induced by RANKL, PMT or PMT in the presence of rapamycin was quantified by determining the pit area. ( f ) Cellular apoptosis was determined by performing a caspase-Glo 3/7 Assay. Cells were incubated for 48 h with PMT (5 nM), the solvent control DMSO, rapamycin (10 ng/ml) or left untreated. As a positive control for apoptosis, cells were incubated with 1 μM doxorubicin for 6 h. Cells were lysed and the luminescence signal was recorded. The indicated standard deviations of all figures were calculated from three independent experiments (mean ± SD; n = 3). Statistical analysis for all graphs was performed by unpaired Student’s t -test (*: p ≤ 0.05, **: p ≤0.005, ***: p ≤ 0.0005****: p ≤ 0.0001)

    Journal: Cell Communication and Signaling : CCS

    Article Title: Pasteurella multocida toxin- induced osteoclastogenesis requires mTOR activation

    doi: 10.1186/s12964-015-0117-7

    Figure Lengend Snippet: PMT-stimulated mTOR activation is required for the full functional activity of the osteoclasts. ( a ) Lysates of cells stimulated for 4 days with PMT concentrations ranging from 10 pM to 5 nM were analyzed for activity of cathepsin K (cleavage of its specific substrate) by fluorescence detection. ( b ) RAW264.7 cells stimulated with PMT (5 nM) w or w/o rapamycin (10 ng/ml) were analyzed for cathepsin K activity by fluorescence detection. As controls, cells were treated with rapamycin, the solvent control DMSO or were left untreated. ( c ) Cells were plated and stimulated on fluoresceinated calcium phosphate plates for 6 days to allow osteoclast differentiation. A fluorescent dye released into the supernatant dependent on the degree of bone resorption was measured with a fluorometer. ( d ) Osteoclasts were differentiated with RANKL and PMT, respectively, on bone slices and the pit area of resorbed bone was determined. ( e ) Bone resorption induced by RANKL, PMT or PMT in the presence of rapamycin was quantified by determining the pit area. ( f ) Cellular apoptosis was determined by performing a caspase-Glo 3/7 Assay. Cells were incubated for 48 h with PMT (5 nM), the solvent control DMSO, rapamycin (10 ng/ml) or left untreated. As a positive control for apoptosis, cells were incubated with 1 μM doxorubicin for 6 h. Cells were lysed and the luminescence signal was recorded. The indicated standard deviations of all figures were calculated from three independent experiments (mean ± SD; n = 3). Statistical analysis for all graphs was performed by unpaired Student’s t -test (*: p ≤ 0.05, **: p ≤0.005, ***: p ≤ 0.0005****: p ≤ 0.0001)

    Article Snippet: The mTOR Inhibitor I (rapamycin) was purchased from Calbiochem and used at 10 ng/ml.

    Techniques: Activation Assay, Functional Assay, Activity Assay, Fluorescence, Caspase-Glo Assay, Incubation, Positive Control

    Inhibition of mTOR-p70 S6K pathway by rapamycin does not affect 5-MCDE–induced AP-1 transactivation. 8 × 10 3 P + 1-1 cells were seeded into each well of a 96-well plate. After being cultured at 37°C overnight, cells were pretreated with 20 μM rapamycin for 30 min and exposed to 1 μM 5-MCDE for various time points as indicated. The luciferase activity was measured and the results are presented as relative AP-1 activity. Each bar indicates the mean and SD of four repeat assay wells.

    Journal: The Journal of Cell Biology

    Article Title: PI-3K and Akt are mediators of AP-1 induction by 5-MCDE in mouse epidermal Cl41 cells

    doi: 10.1083/jcb.200401004

    Figure Lengend Snippet: Inhibition of mTOR-p70 S6K pathway by rapamycin does not affect 5-MCDE–induced AP-1 transactivation. 8 × 10 3 P + 1-1 cells were seeded into each well of a 96-well plate. After being cultured at 37°C overnight, cells were pretreated with 20 μM rapamycin for 30 min and exposed to 1 μM 5-MCDE for various time points as indicated. The luciferase activity was measured and the results are presented as relative AP-1 activity. Each bar indicates the mean and SD of four repeat assay wells.

    Article Snippet: The PI-3K inhibitors, wortmannin, and Ly294002 and the mTOR pathway inhibitor rapamycin were procured from Calbiochem.

    Techniques: Inhibition, Cell Culture, Luciferase, Activity Assay

    (A) Effect of mTOR inhibitors on PCSK9 mRNA expression. Five selected resistant and sensitive Lymphoblastoid Cell Lines (LCLs) for mTOR inhibitors were treated with sirolimus (0.2 μM), everolimus (0.33 μM) or DMSO for 3 d. QRT-PCR was performed to measure the expression of PCSK9 pre and post drug treatment. (B) Representative Western blot for protein expression. Western blot analysis with antibodies against mTOR, phosphorylated Ser2448mTOR, PCSK9 was performed using protein lysate from cells treated under the same conditions as those in (A). Actin was used as a loading control. (C) Quantification of relative protein levels for all 5 LCLs. Results were calculated with Grey-scan value. * p

    Journal: Journal of cardiovascular translational research

    Article Title: Sirolimus therapy is associated with elevation in circulating PCSK9 levels in cardiac transplant patients

    doi: 10.1007/s12265-016-9719-8

    Figure Lengend Snippet: (A) Effect of mTOR inhibitors on PCSK9 mRNA expression. Five selected resistant and sensitive Lymphoblastoid Cell Lines (LCLs) for mTOR inhibitors were treated with sirolimus (0.2 μM), everolimus (0.33 μM) or DMSO for 3 d. QRT-PCR was performed to measure the expression of PCSK9 pre and post drug treatment. (B) Representative Western blot for protein expression. Western blot analysis with antibodies against mTOR, phosphorylated Ser2448mTOR, PCSK9 was performed using protein lysate from cells treated under the same conditions as those in (A). Actin was used as a loading control. (C) Quantification of relative protein levels for all 5 LCLs. Results were calculated with Grey-scan value. * p

    Article Snippet: Cytotoxicity data for mTOR inhibitor sirolimus (Sigma, St. Louis, MO) and everolimus (Sigma, St. Louis, MO) were performed in all LCLs as described before [ ].

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    Growth and cell cycle regulation during endothelial differentiation and rapamycin treatment . a) Rate of cell division. RoSH2 cells were labeled with CFDA, a cell-permeant fluorescent dye, cultured for 24 hours and re-plated on gelatin-coated plates to be maintained as undifferentiated cells (Undif) or on matrigel to induce differentiation (Dif). Cells were harvested at 0, 24, 48 and 72 hours. At 48 hours after replating, half of the remaining plates of cells under undifferentiating condition or differentiating condition were treated with 50 ηM rapamycin (R undif and Rdif, respectively). Median cellular fluorescence of the harvested cells was measured by flow cytometry and the number of cell divisions was calculated as a function of the loss in fluorescence; b) Cell cycle progression during endothelial differentiation. RoSH2 cells were plated on either gelatin-coated plate (self-renewing) or matrigel (differentiating) and labeled with BrdU for 16 hours. After removing BrdU, half of the gelatin-coated plates were treated with 50 ηM rapamycin. At 0, 6 and 12 hours, cells were harvested, stained with anti-BrdU and PI. DNA content of BrdU-labeled cells as measured by PI was analyzed by flow cytometry.

    Journal: Journal of Molecular Signaling

    Article Title: PI3 K/Akt/mTOR-mediated translational control regulates proliferation and differentiation of lineage-restricted RoSH stem cell lines

    doi: 10.1186/1750-2187-2-9

    Figure Lengend Snippet: Growth and cell cycle regulation during endothelial differentiation and rapamycin treatment . a) Rate of cell division. RoSH2 cells were labeled with CFDA, a cell-permeant fluorescent dye, cultured for 24 hours and re-plated on gelatin-coated plates to be maintained as undifferentiated cells (Undif) or on matrigel to induce differentiation (Dif). Cells were harvested at 0, 24, 48 and 72 hours. At 48 hours after replating, half of the remaining plates of cells under undifferentiating condition or differentiating condition were treated with 50 ηM rapamycin (R undif and Rdif, respectively). Median cellular fluorescence of the harvested cells was measured by flow cytometry and the number of cell divisions was calculated as a function of the loss in fluorescence; b) Cell cycle progression during endothelial differentiation. RoSH2 cells were plated on either gelatin-coated plate (self-renewing) or matrigel (differentiating) and labeled with BrdU for 16 hours. After removing BrdU, half of the gelatin-coated plates were treated with 50 ηM rapamycin. At 0, 6 and 12 hours, cells were harvested, stained with anti-BrdU and PI. DNA content of BrdU-labeled cells as measured by PI was analyzed by flow cytometry.

    Article Snippet: To test the effects of PI3 K inhibitors, LY294002 and wortmannin on mTOR activity, cells were cultured for 24 h immediately after passaging and then deprived of serum for 20 h. Cells were then treated with or without 50 μM LY294002 (Sigma, St. Louis, MO) or 100 ηM wortmannin (Sigma, St. Louis, MO) for 15 min and then stimulated with 100 ηM insulin for 20 min. For other treatment, 80% confluent cultures were treated with 50 ηM rapamycin (Sigma, St. Louis, MO), 10 μM Ras inhibitor FTase inhibitor III (Calbiochen-Novabrochem Corp, La Jolla, CA) and 50 μM MEK inhibitor PD98059 (Calbiochen-Novabrochem Corp, La Jolla, CA).

    Techniques: Labeling, Cell Culture, Fluorescence, Flow Cytometry, Cytometry, Staining

    Phosphorylation of mTOR targets and expression of endothelial markers during differentiation and rapamycin treatment . At 0, 6, 24 and 48 hours after RoSH2 cells were induced to differentiate by plating on matrigel-coated plates or treated with 50 ηM rapamycin, the cells were harvested for western blot analysis and probed for a, d) endothelial markers, Tie-2 and Flk-1; b, c) Eif4ebp1 and phosphorylated Rps6kb1. Tsc2 protein was used as an internal control for loading between lanes and between blots.

    Journal: Journal of Molecular Signaling

    Article Title: PI3 K/Akt/mTOR-mediated translational control regulates proliferation and differentiation of lineage-restricted RoSH stem cell lines

    doi: 10.1186/1750-2187-2-9

    Figure Lengend Snippet: Phosphorylation of mTOR targets and expression of endothelial markers during differentiation and rapamycin treatment . At 0, 6, 24 and 48 hours after RoSH2 cells were induced to differentiate by plating on matrigel-coated plates or treated with 50 ηM rapamycin, the cells were harvested for western blot analysis and probed for a, d) endothelial markers, Tie-2 and Flk-1; b, c) Eif4ebp1 and phosphorylated Rps6kb1. Tsc2 protein was used as an internal control for loading between lanes and between blots.

    Article Snippet: To test the effects of PI3 K inhibitors, LY294002 and wortmannin on mTOR activity, cells were cultured for 24 h immediately after passaging and then deprived of serum for 20 h. Cells were then treated with or without 50 μM LY294002 (Sigma, St. Louis, MO) or 100 ηM wortmannin (Sigma, St. Louis, MO) for 15 min and then stimulated with 100 ηM insulin for 20 min. For other treatment, 80% confluent cultures were treated with 50 ηM rapamycin (Sigma, St. Louis, MO), 10 μM Ras inhibitor FTase inhibitor III (Calbiochen-Novabrochem Corp, La Jolla, CA) and 50 μM MEK inhibitor PD98059 (Calbiochen-Novabrochem Corp, La Jolla, CA).

    Techniques: Expressing, Western Blot

    Translational control during endothelial differentiation and rapamycin treatment . a) Preparation of total RNA and ribosome-enriched RNA. RoSH2 cells, RoSH2 induced to differentiate by plating on matrigel for 48 hours and RoSH2 cells treated with 50 ηM rapamycin for 48 hours were harvested. Total and ribosome-enriched RNAs were quantitated by absorbance at 260 nm. 2 μg of RNA from each of the two cellular fractions were separated on a 1.0% agarose gel, stained with ethidium bromide and visualized under UV illumination. Top panel Representative RNA samples from total and ribosome-enriched RNAs prepared from undifferentiated (Undiff), differentiated (Diff) and rapamycin-treated (Rapa) RoSH2 cells are shown. Bottom panel For each sample, 1 μl of serially diluted RNA sample mixed with 1 μl of 0.5 ηg/μl ethidium bromide was visualized under UV illumination to verify RNA loading in each lane; RNA was isolated from total cellular extract (total) and ribosome-enriched subcellular fraction (ribo) prepared from RoSH2 cells before and 48 hours after induction of endothelial differentiation by plating matrigel-coated plates; b) Distribution of rpL5 mRNA in total and ribosome-enriched RNAs before and after differentiation. RT-PCR using oligo-dT-primed cDNA, and Rpl5 and Fkbp12 specific primers was performed on 10 fold serial dilution i.e. 1×, 10× and 100× of RNAs.; c) RT-PCR analysis of transcript abundance in RoSH2 cells before and after differentiation. RoSH2 cells were induced to undergo endothelial differentiation by plating on matrigel. Total and ribosome-enriched RNA were purified at 0 and 48 hours and analyzed by RT-PCR for transcript abundance of G1 cyclins and endothelial receptors; d) Western blot analysis. RoSH2 cells were induced to undergo endothelial differentiation by plating on matrigel and at 0, 6, 24 and 48 hours, cell lysates were prepared and assayed by western blot analysis. Tsc2 protein was used as an internal control for loading between lanes and between blots; e) Effect of rapamycin on Rpl5, cyclinD2 and Tek transcript abundance in total and ribosome-enriched RNA. Total and ribosome-enriched RNA were isolated from RoSH2 cells treated for 0 and 48 hours with 50 ηM rapamycin treatment and analyzed by RT-PCR; f) Western blot analysis. Cell lysates from rapamycin-treated RoSH2 cells at 0, 6, 24 and 48 hours were assayed by western blot analysis for cyclinD2 and Tek. Tsc2 protein was used as an internal control for loading between lanes and between blots.

    Journal: Journal of Molecular Signaling

    Article Title: PI3 K/Akt/mTOR-mediated translational control regulates proliferation and differentiation of lineage-restricted RoSH stem cell lines

    doi: 10.1186/1750-2187-2-9

    Figure Lengend Snippet: Translational control during endothelial differentiation and rapamycin treatment . a) Preparation of total RNA and ribosome-enriched RNA. RoSH2 cells, RoSH2 induced to differentiate by plating on matrigel for 48 hours and RoSH2 cells treated with 50 ηM rapamycin for 48 hours were harvested. Total and ribosome-enriched RNAs were quantitated by absorbance at 260 nm. 2 μg of RNA from each of the two cellular fractions were separated on a 1.0% agarose gel, stained with ethidium bromide and visualized under UV illumination. Top panel Representative RNA samples from total and ribosome-enriched RNAs prepared from undifferentiated (Undiff), differentiated (Diff) and rapamycin-treated (Rapa) RoSH2 cells are shown. Bottom panel For each sample, 1 μl of serially diluted RNA sample mixed with 1 μl of 0.5 ηg/μl ethidium bromide was visualized under UV illumination to verify RNA loading in each lane; RNA was isolated from total cellular extract (total) and ribosome-enriched subcellular fraction (ribo) prepared from RoSH2 cells before and 48 hours after induction of endothelial differentiation by plating matrigel-coated plates; b) Distribution of rpL5 mRNA in total and ribosome-enriched RNAs before and after differentiation. RT-PCR using oligo-dT-primed cDNA, and Rpl5 and Fkbp12 specific primers was performed on 10 fold serial dilution i.e. 1×, 10× and 100× of RNAs.; c) RT-PCR analysis of transcript abundance in RoSH2 cells before and after differentiation. RoSH2 cells were induced to undergo endothelial differentiation by plating on matrigel. Total and ribosome-enriched RNA were purified at 0 and 48 hours and analyzed by RT-PCR for transcript abundance of G1 cyclins and endothelial receptors; d) Western blot analysis. RoSH2 cells were induced to undergo endothelial differentiation by plating on matrigel and at 0, 6, 24 and 48 hours, cell lysates were prepared and assayed by western blot analysis. Tsc2 protein was used as an internal control for loading between lanes and between blots; e) Effect of rapamycin on Rpl5, cyclinD2 and Tek transcript abundance in total and ribosome-enriched RNA. Total and ribosome-enriched RNA were isolated from RoSH2 cells treated for 0 and 48 hours with 50 ηM rapamycin treatment and analyzed by RT-PCR; f) Western blot analysis. Cell lysates from rapamycin-treated RoSH2 cells at 0, 6, 24 and 48 hours were assayed by western blot analysis for cyclinD2 and Tek. Tsc2 protein was used as an internal control for loading between lanes and between blots.

    Article Snippet: To test the effects of PI3 K inhibitors, LY294002 and wortmannin on mTOR activity, cells were cultured for 24 h immediately after passaging and then deprived of serum for 20 h. Cells were then treated with or without 50 μM LY294002 (Sigma, St. Louis, MO) or 100 ηM wortmannin (Sigma, St. Louis, MO) for 15 min and then stimulated with 100 ηM insulin for 20 min. For other treatment, 80% confluent cultures were treated with 50 ηM rapamycin (Sigma, St. Louis, MO), 10 μM Ras inhibitor FTase inhibitor III (Calbiochen-Novabrochem Corp, La Jolla, CA) and 50 μM MEK inhibitor PD98059 (Calbiochen-Novabrochem Corp, La Jolla, CA).

    Techniques: Agarose Gel Electrophoresis, Staining, Isolation, Reverse Transcription Polymerase Chain Reaction, Serial Dilution, Purification, Western Blot

    Immunoblot analysis of (A) Hela, (B) Caki-1 and (C) Hepa-1 cells. Cells were exposed to either normoxia (N, 21% O 2 ), hypoxia (H, 1% O 2 ) or DP (Dipyridyl, 100µM) treatment for 6 or 24 hours. 30µg of total cell lysate was loaded and western blotting was performed for the following proteins: HIF-1α, HIF-2α, p70S6K-P (Thr 389), p70S6K, rpS6-P (Ser 235/236), rpS6 and ß-actin. Hepa-1 cells are either wildtype cells (Hepa-1 C1C7) or HIF-1ß deficient (Hepa-1 C4). Therefore, these cells were additionally analyzed for the expression of HIF-1ß. The mTOR inhibitor rapamycin was applied 45 minutes prior to hypoxic/pharmacological exposure at a concentration of 100nM.

    Journal: Journal of Cancer

    Article Title: HIF is not essential for suppression of experimental tumor growth by mTOR inhibition

    doi: 10.7150/jca.16486

    Figure Lengend Snippet: Immunoblot analysis of (A) Hela, (B) Caki-1 and (C) Hepa-1 cells. Cells were exposed to either normoxia (N, 21% O 2 ), hypoxia (H, 1% O 2 ) or DP (Dipyridyl, 100µM) treatment for 6 or 24 hours. 30µg of total cell lysate was loaded and western blotting was performed for the following proteins: HIF-1α, HIF-2α, p70S6K-P (Thr 389), p70S6K, rpS6-P (Ser 235/236), rpS6 and ß-actin. Hepa-1 cells are either wildtype cells (Hepa-1 C1C7) or HIF-1ß deficient (Hepa-1 C4). Therefore, these cells were additionally analyzed for the expression of HIF-1ß. The mTOR inhibitor rapamycin was applied 45 minutes prior to hypoxic/pharmacological exposure at a concentration of 100nM.

    Article Snippet: Protein Extraction and Immunoblotting For protein extraction, cells were seeded 24 h before experiments and then pretreated for 45 min with the mTOR inhibitor rapamycin (100nM) (Sigma-Aldrich) and subsequently subjected to 6 h or 24 h of stimulation.

    Techniques: Western Blot, Expressing, Concentration Assay

    Increased S6K1-activity in eIF3b and/or eIF3c-depleted cells A. Western blot analysis of eIF3b and Cyclin D1 was performed in eIF3b and/or eIF3c-depleted IMR-90 cells at 16h after re-plating. B. At the same time-point, eIF3c, p27 and mTOR expression levels were determined in eIF3b and/or eIF3c-depleted IMR-90 cells. To avoid any interference in the detection of eIF3b and eIF3c due to similar protein size on the same membrane, same lysates were detected on separate membranes. C. Expression levels of mTORC1-specific targets were evaluated by western blotting in eIF3b/c- or mTOR-depleted IMR-90 cells. D. mTORC2-specific target p-AKT (S473) as well as PDCD4 expression levels were detected in cell lysates of IMR-90 as indicated. E. mTORC1 and mTORC2-specific targets were determined in HEK293 cells by immunoblotting. F. 48 hours post transfection, IMR-90 cells were treated with 100nM rapamycin or DMSO for another 24 hours. Total and phosphorylated S6K1 and S6 protein levels were evaluated by immunoblotting. αTubulin was used as appropriate loading control in all panels.

    Journal: Oncotarget

    Article Title: eIF3 controls cell size independently of S6K1-activity

    doi:

    Figure Lengend Snippet: Increased S6K1-activity in eIF3b and/or eIF3c-depleted cells A. Western blot analysis of eIF3b and Cyclin D1 was performed in eIF3b and/or eIF3c-depleted IMR-90 cells at 16h after re-plating. B. At the same time-point, eIF3c, p27 and mTOR expression levels were determined in eIF3b and/or eIF3c-depleted IMR-90 cells. To avoid any interference in the detection of eIF3b and eIF3c due to similar protein size on the same membrane, same lysates were detected on separate membranes. C. Expression levels of mTORC1-specific targets were evaluated by western blotting in eIF3b/c- or mTOR-depleted IMR-90 cells. D. mTORC2-specific target p-AKT (S473) as well as PDCD4 expression levels were detected in cell lysates of IMR-90 as indicated. E. mTORC1 and mTORC2-specific targets were determined in HEK293 cells by immunoblotting. F. 48 hours post transfection, IMR-90 cells were treated with 100nM rapamycin or DMSO for another 24 hours. Total and phosphorylated S6K1 and S6 protein levels were evaluated by immunoblotting. αTubulin was used as appropriate loading control in all panels.

    Article Snippet: Inhibitor treatments Cycloheximide (Calbiochem, #239764) was added to the cells for 3, 5 hours at a final concentration of 50 μM to inhibit protein synthesis. mTOR specific inhibitor rapamycin (Calbiochem, #553211) was used at a final concentration of 100 nM.

    Techniques: Activity Assay, Western Blot, Expressing, Transfection

    TOR signaling is required for early trypsin protein synthesis in amino acid fed mosquitoes. A) Injection of TOR dsRNA had no effect on early trypsin transcript levels in unfed mosquitoes based on quantitative RT-PCR. B) Injection of TOR dsRNA or rapamycin

    Journal:

    Article Title: TOR Signaling is Required for Amino Acid Stimulation of Early Trypsin Protein Synthesis in the Midgut of Aedes aegypti Mosquitoes

    doi: 10.1016/j.ibmb.2008.07.003

    Figure Lengend Snippet: TOR signaling is required for early trypsin protein synthesis in amino acid fed mosquitoes. A) Injection of TOR dsRNA had no effect on early trypsin transcript levels in unfed mosquitoes based on quantitative RT-PCR. B) Injection of TOR dsRNA or rapamycin

    Article Snippet: For the rapamycin experiments, midguts were pre-treated at 30°C in a humid chamber for 1 hour with or without the TOR inhibitor rapamycin (EMD Biosciences, La Jolla, CA) at various concentrations as described in .

    Techniques: Injection, Quantitative RT-PCR

    IGF-I dependent DDR1 protein upregulation is downstream the PI3K/AKT pathway and does not require activation of the ERK1/2 and the mTOR pathways MCF-7 cells grown with serum stripped 2.5% of FCS for 24 h, were pretreated with various kinase inhibitors at the indicated doses for 1 h. Cells were then stimulated with 50 nM IGF-I for 24 h, lysed and analyzed by western blotting to evaluate DDR1 protein expression. ( A ) Cells treated with the PI3K inhibitor LY294002 (LY), and ( B ) the AKT inhibitor1/2 (iAKT). ( C ) Cells treated with the MEK1 inhibitor U0126, and ( D ) with the TORC1 inhibitor Rapamycin (Rapa). Immunoblot for α-tubulin was used as control for protein loading. Each blot shown is representative of three independent experiments. Values are mean ± SEM of three separate experiments. * P

    Journal: Oncotarget

    Article Title: IGF-I induces upregulation of DDR1 collagen receptor in breast cancer cells by suppressing MIR-199a-5p through the PI3K/AKT pathway

    doi: 10.18632/oncotarget.6524

    Figure Lengend Snippet: IGF-I dependent DDR1 protein upregulation is downstream the PI3K/AKT pathway and does not require activation of the ERK1/2 and the mTOR pathways MCF-7 cells grown with serum stripped 2.5% of FCS for 24 h, were pretreated with various kinase inhibitors at the indicated doses for 1 h. Cells were then stimulated with 50 nM IGF-I for 24 h, lysed and analyzed by western blotting to evaluate DDR1 protein expression. ( A ) Cells treated with the PI3K inhibitor LY294002 (LY), and ( B ) the AKT inhibitor1/2 (iAKT). ( C ) Cells treated with the MEK1 inhibitor U0126, and ( D ) with the TORC1 inhibitor Rapamycin (Rapa). Immunoblot for α-tubulin was used as control for protein loading. Each blot shown is representative of three independent experiments. Values are mean ± SEM of three separate experiments. * P

    Article Snippet: The following kinase inhibitors were used: the IGF-IR inhibitor NVP-AEW541 (Cayman Chemical, Ann Arbor, USA); the PI3 kinase inhibitor LY 294002 (Calbiochem, Merck Millipore, Nottingham, UK); the MEK1 inhibitor U0126 (Sigma-Aldrich, Saint Louis Missouri, USA); the TORC1 inhibitor rapamycin (Sigma-Aldrich); the AKT 1,2 inhibitor (Sigma-Aldrich, Saint Louis Missouri, USA).

    Techniques: Activation Assay, Western Blot, Expressing

    Signaling pathways regulated by liraglutide. The molecular pathways shown in gray boxes illustrate our novel observations. Akt: Protein kinase B (PKB), AMPAR: α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, BDNF: brain-derived neurotrophic factor, ERK: extracellular signal-regulated kinase, GLP-1R: glucagon-like peptide 1 receptor, GluA1: AMPA receptor subunit GluR1, mTORC1: mammalian target of rapamycin complex 1, NBQX: 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, p70S6K: P70S6 kinase, PI3K: Phosphoinositide 3-kinase, PSD-95: Post Synaptic Density 95 protein, TrKBR: tropomyosin receptor kinase B receptor, 4E-BP-1: eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1.

    Journal: Frontiers in Neuroscience

    Article Title: Liraglutide Activates mTORC1 Signaling and AMPA Receptors in Rat Hippocampal Neurons Under Toxic Conditions

    doi: 10.3389/fnins.2018.00756

    Figure Lengend Snippet: Signaling pathways regulated by liraglutide. The molecular pathways shown in gray boxes illustrate our novel observations. Akt: Protein kinase B (PKB), AMPAR: α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, BDNF: brain-derived neurotrophic factor, ERK: extracellular signal-regulated kinase, GLP-1R: glucagon-like peptide 1 receptor, GluA1: AMPA receptor subunit GluR1, mTORC1: mammalian target of rapamycin complex 1, NBQX: 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, p70S6K: P70S6 kinase, PI3K: Phosphoinositide 3-kinase, PSD-95: Post Synaptic Density 95 protein, TrKBR: tropomyosin receptor kinase B receptor, 4E-BP-1: eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1.

    Article Snippet: Specific kinase inhibitors were purchased from the following sources: PI3K inhibitor LY294002 from Cell Signaling Technology; MEK inhibitor PD98059 and mTORC1 inhibitor rapamycin from Calbiochem (San Diego, CA, United States); and the AMPA receptor inhibitor 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f] quinoxaline-7-sulfonamide (NBQX) from Tocris Bioscience (Ballwin, MO, United States).

    Techniques: Derivative Assay, Binding Assay

    mTORC1 inhibition does not induce premature cell death during HCMV infection. (A) HFFs were infected with AD wt at an MOI of 1 in the presence or absence of rapamycin (10 nM) or torin 1 (250 nM). The morphology of infected cells was analyzed with a phase-contrast microscope at 72 hpi. (B) HFFs were either pretreated with rapamycin for 6 h and then infected with AD wt in the presence of rapamycin, or infected with AD wt in the presence of torin 1. At 72 hpi, infected cells were collected and analyzed for cell death (PARP cleavage) and mTORC1 activation (phosphorylation of S6K and 4E-BP1). HFFs infected with AD pm UL38 were included as the control for mutant virus-induced cell death.

    Journal: Journal of Virology

    Article Title: The Human Cytomegalovirus Protein pUL38 Suppresses Endoplasmic Reticulum Stress-Mediated Cell Death Independently of Its Ability To Induce mTORC1 Activation ▿

    doi: 10.1128/JVI.00572-11

    Figure Lengend Snippet: mTORC1 inhibition does not induce premature cell death during HCMV infection. (A) HFFs were infected with AD wt at an MOI of 1 in the presence or absence of rapamycin (10 nM) or torin 1 (250 nM). The morphology of infected cells was analyzed with a phase-contrast microscope at 72 hpi. (B) HFFs were either pretreated with rapamycin for 6 h and then infected with AD wt in the presence of rapamycin, or infected with AD wt in the presence of torin 1. At 72 hpi, infected cells were collected and analyzed for cell death (PARP cleavage) and mTORC1 activation (phosphorylation of S6K and 4E-BP1). HFFs infected with AD pm UL38 were included as the control for mutant virus-induced cell death.

    Article Snippet: Pharmaceutical agents used in the present study included the ER stress-inducing agent tunicamycin (Sigma) and mTORC1 inhibitors rapamycin (Sigma) and torin 1 (a generous gift from David Sabatini at Whitehead Institute for Biomedical Research).

    Techniques: Inhibition, Infection, Microscopy, Activation Assay, Mutagenesis

    (A) Schematic diagram of the ER stress response. Three sensors on the ER membrane, PERK, ATF6, and IRE1, are activated upon ER stress. Activated PERK phosphorylates eIF-2α to attenuate global protein translation but selectively enhance ATF4 translation. Activated ATF6 is cleaved and translocated to the nucleus, where it upregulates ER chaperone expression. Once activated, the endoribonuclease activity of IRE1 mediates splicing of Xbp-1, which encodes a transcriptional activator to induce transcription of ERAD components. The kinase activity of IRE1 phosphorylates JNK, which can ultimately lead to cell death. Viral protein pUL38 activates PERK-dependent ATF4 expression, suppresses persistent JNK phosphorylation, and inhibits ER stress-induced cell death. Modulation of other components of the ER stress response by HCMV is described in the text. (B) Schematic diagram of the mTOR pathway. Growth factors and metabolic stresses regulate mTORC1 activation by acting on the TSC1/2 complex. The TSC1/2 complex functions as a GTPase-activating protein for Rheb, which activates mTORC1 in its GTP-bound form. Once activated, mTORC1 phosphorylates 4E-BP1 and S6K, two well-characterized substrates regulating protein translation. Amino acids activate mTORC1 through a parallel pathway independent of TSC1/2 or Rheb. pUL38 binds to TSC2 and activates mTORC1. Rapamycin inhibits mTORC1 by binding to mTOR as a rapamycin-FKBP12 complex to interfere with its substrate binding capability. Torin 1 directly inactivates mTOR kinase activity by competing with ATP binding.

    Journal: Journal of Virology

    Article Title: The Human Cytomegalovirus Protein pUL38 Suppresses Endoplasmic Reticulum Stress-Mediated Cell Death Independently of Its Ability To Induce mTORC1 Activation ▿

    doi: 10.1128/JVI.00572-11

    Figure Lengend Snippet: (A) Schematic diagram of the ER stress response. Three sensors on the ER membrane, PERK, ATF6, and IRE1, are activated upon ER stress. Activated PERK phosphorylates eIF-2α to attenuate global protein translation but selectively enhance ATF4 translation. Activated ATF6 is cleaved and translocated to the nucleus, where it upregulates ER chaperone expression. Once activated, the endoribonuclease activity of IRE1 mediates splicing of Xbp-1, which encodes a transcriptional activator to induce transcription of ERAD components. The kinase activity of IRE1 phosphorylates JNK, which can ultimately lead to cell death. Viral protein pUL38 activates PERK-dependent ATF4 expression, suppresses persistent JNK phosphorylation, and inhibits ER stress-induced cell death. Modulation of other components of the ER stress response by HCMV is described in the text. (B) Schematic diagram of the mTOR pathway. Growth factors and metabolic stresses regulate mTORC1 activation by acting on the TSC1/2 complex. The TSC1/2 complex functions as a GTPase-activating protein for Rheb, which activates mTORC1 in its GTP-bound form. Once activated, mTORC1 phosphorylates 4E-BP1 and S6K, two well-characterized substrates regulating protein translation. Amino acids activate mTORC1 through a parallel pathway independent of TSC1/2 or Rheb. pUL38 binds to TSC2 and activates mTORC1. Rapamycin inhibits mTORC1 by binding to mTOR as a rapamycin-FKBP12 complex to interfere with its substrate binding capability. Torin 1 directly inactivates mTOR kinase activity by competing with ATP binding.

    Article Snippet: Pharmaceutical agents used in the present study included the ER stress-inducing agent tunicamycin (Sigma) and mTORC1 inhibitors rapamycin (Sigma) and torin 1 (a generous gift from David Sabatini at Whitehead Institute for Biomedical Research).

    Techniques: Expressing, Activity Assay, Activation Assay, Binding Assay

    Inhibition of mTORC1 does not compromise the ability of pUL38 to block ER stress-induced cell death. (A) pUL38 1–239 inhibits tunicamycin (TM)-induced cell death. Confluent HFFs expressing the empty vector, pUL38, or pUL38 1–239 were treated with 2 μg/ml tunicamycin at 36 h after seeding and stained with fluorescein-conjugated annexin V and TO-PRO3 at 72 h after treatment. The percentage of annexin V- and/or TO-PRO3-positive cells was determined by flow cytometry. PARP cleavage and S6K phosphorylation were analyzed by immunoblotting. (B) Inhibition of mTORC1 by rapamycin (rap) does not compromise the ability of pUL38 to inhibit tunicamycin-induced cell death. HFFs expressing the empty vector or pUL38 were treated with rapamycin (10 nM) only or tunicamycin only (2 μg/ml) or pretreated with rapamycin for 6 h and subsequently treated with rapamycin and tunicamycin. At 72 h after treatment, the percentage of annexin V- and/or TO-PRO3-positive cells, PARP cleavage, and S6K phosphorylation were determined as described for panel A. (C) Inhibition of mTORC1 by torin 1 does not compromise the ability of pUL38 to inhibit tunicamycin-induced cell death. HFFs expressing the empty vector or pUL38 were treated with torin 1 (250 nM) only, tunicamycin only (2 μg/ml), or torin 1 and tunicamycin. At 72 h after treatment, the percentage of annexin V- and/or TO-PRO3-positive cells, PARP cleavage, and S6K phosphorylation were determined as described for panel A.

    Journal: Journal of Virology

    Article Title: The Human Cytomegalovirus Protein pUL38 Suppresses Endoplasmic Reticulum Stress-Mediated Cell Death Independently of Its Ability To Induce mTORC1 Activation ▿

    doi: 10.1128/JVI.00572-11

    Figure Lengend Snippet: Inhibition of mTORC1 does not compromise the ability of pUL38 to block ER stress-induced cell death. (A) pUL38 1–239 inhibits tunicamycin (TM)-induced cell death. Confluent HFFs expressing the empty vector, pUL38, or pUL38 1–239 were treated with 2 μg/ml tunicamycin at 36 h after seeding and stained with fluorescein-conjugated annexin V and TO-PRO3 at 72 h after treatment. The percentage of annexin V- and/or TO-PRO3-positive cells was determined by flow cytometry. PARP cleavage and S6K phosphorylation were analyzed by immunoblotting. (B) Inhibition of mTORC1 by rapamycin (rap) does not compromise the ability of pUL38 to inhibit tunicamycin-induced cell death. HFFs expressing the empty vector or pUL38 were treated with rapamycin (10 nM) only or tunicamycin only (2 μg/ml) or pretreated with rapamycin for 6 h and subsequently treated with rapamycin and tunicamycin. At 72 h after treatment, the percentage of annexin V- and/or TO-PRO3-positive cells, PARP cleavage, and S6K phosphorylation were determined as described for panel A. (C) Inhibition of mTORC1 by torin 1 does not compromise the ability of pUL38 to inhibit tunicamycin-induced cell death. HFFs expressing the empty vector or pUL38 were treated with torin 1 (250 nM) only, tunicamycin only (2 μg/ml), or torin 1 and tunicamycin. At 72 h after treatment, the percentage of annexin V- and/or TO-PRO3-positive cells, PARP cleavage, and S6K phosphorylation were determined as described for panel A.

    Article Snippet: Pharmaceutical agents used in the present study included the ER stress-inducing agent tunicamycin (Sigma) and mTORC1 inhibitors rapamycin (Sigma) and torin 1 (a generous gift from David Sabatini at Whitehead Institute for Biomedical Research).

    Techniques: Inhibition, Blocking Assay, Expressing, Plasmid Preparation, Staining, Flow Cytometry, Cytometry

    Induction of drug resistance in HMEC-1 cells and sunitinib-induced activation of autophagy. (A) Replication times of cultured HMEC-1 cells in the presence of indicated concentrations of sunitinib. Data were calculated from more than two repetitions. (B) Evolution of the IC50 of sunitinib in HMEC-1 cells after treatment with 6 µ M sunitinib for different time periods. (C) Plotted survival rates of HMEC-1 cells and sunitinib-resistant HMEC 6 µ M cells. Normal HMEC-1 cells and sunitinib-resistant HMEC 6 µ M cells were exposed to the indicated concentrations of sunitinib for 72 h, and the cells were trypsinized and counted. Non-treated cells were set as 100%. (A-C) Curves are derived from the means of more than three experiments. (D) LysoTracker DND-99 density of HMEC-1 cells following incubation with 6 µ M sunitinib for different time periods. Cells were treated with sunitinib for 24 or 48 h and LysoTracker DND-99 was added followed by flow cytometric analysis. (E) LysoTracker DND-99 density of sunitinib-treated HMEC-1 cells and HMEC 6 µ M cells. The cells were washed five times and then incubated with LysoTracker DND-99. Fluorescence was measured with the control cells without LysoTracker DND-99 staining using flow cytometry. (F-H) Western blot analysis using anti-LAMP-1, anti-LC3 and anti-p62 antibodies. HMEC-1 and HMEC 6 µ M cells were exposed to 6 µ M sunitinib, or autophagy inducer 5 µ M rapamycin, or Hanks' media for 24 h. Subsequently, the cells were collected and extracted proteins were used for western blotting. The experiments were repeated at least three times. * P

    Journal: International Journal of Oncology

    Article Title: Drug resistance-related sunitinib sequestration in autophagolysosomes of endothelial cells

    doi: 10.3892/ijo.2019.4924

    Figure Lengend Snippet: Induction of drug resistance in HMEC-1 cells and sunitinib-induced activation of autophagy. (A) Replication times of cultured HMEC-1 cells in the presence of indicated concentrations of sunitinib. Data were calculated from more than two repetitions. (B) Evolution of the IC50 of sunitinib in HMEC-1 cells after treatment with 6 µ M sunitinib for different time periods. (C) Plotted survival rates of HMEC-1 cells and sunitinib-resistant HMEC 6 µ M cells. Normal HMEC-1 cells and sunitinib-resistant HMEC 6 µ M cells were exposed to the indicated concentrations of sunitinib for 72 h, and the cells were trypsinized and counted. Non-treated cells were set as 100%. (A-C) Curves are derived from the means of more than three experiments. (D) LysoTracker DND-99 density of HMEC-1 cells following incubation with 6 µ M sunitinib for different time periods. Cells were treated with sunitinib for 24 or 48 h and LysoTracker DND-99 was added followed by flow cytometric analysis. (E) LysoTracker DND-99 density of sunitinib-treated HMEC-1 cells and HMEC 6 µ M cells. The cells were washed five times and then incubated with LysoTracker DND-99. Fluorescence was measured with the control cells without LysoTracker DND-99 staining using flow cytometry. (F-H) Western blot analysis using anti-LAMP-1, anti-LC3 and anti-p62 antibodies. HMEC-1 and HMEC 6 µ M cells were exposed to 6 µ M sunitinib, or autophagy inducer 5 µ M rapamycin, or Hanks' media for 24 h. Subsequently, the cells were collected and extracted proteins were used for western blotting. The experiments were repeated at least three times. * P

    Article Snippet: Western blot analysis The HMEC-1 cells were treated with or without sunitinib 6 µ M for 24 h, or with the autophagy inducer rapamycin (Sigma-Aldrich; Merck KGaA; cat. no. 553210; 5 µ M) for 8 h, or Hanks' solution (Sigma-Aldrich; Merck KGaA; cat. no. H6648) for 24 h at 37°C.

    Techniques: Activation Assay, Cell Culture, Derivative Assay, Incubation, Flow Cytometry, Fluorescence, Staining, Cytometry, Western Blot

    Immunohistochemical analysis of the effects of LV-Atg7 treatment in α-syn tg mice. For panels A–M, vibratome sections from non tg and α-syn tg mice that received LV injections into the cortex and hippocampus were immunolabeled with antibodies against Atg7 or α-syn and imaged with a digital microscope. Panels A′–M′ represent higher-power images from the hippocampus of the corresponding low-power panels in panels A–M. For panels O–T, effects of rapamycin treatment on α-syn accumulation, autophagy and neuronal integrity in the brains of α-syn tg mice. For panels A–M, vibratome sections from the hippocampus of non tg and α-syn tg mice were immunolabeled with an antibody against MAP2 and imaged with a laser scanning confocal microscope, and images were obtained from the temporal cortex. (A–F) Representative sections from the brains of non tg (A, B) and α-syn tg mice (C–F) that received injections with LV-control (A–D) or LV-Atg7 (E, F) and were immunolabeled with an antibody against Atg7. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (G) Semi-quantitative image analysis of Atg7 immunoreactivity in non tg and α-syn tg mice show increased Atg7 levels ipsilateral to the injection sites in the brains of animals that received LV-Atg7. (H–M) Representative sections from the brains of non tg (H, I) and α-syn tg mice (J–M) that received injections with LV-control (H–K) or LV-Atg7 (L, M) and were immunolabeled with an antibody against α-syn. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (N) Semi-quantitative image analysis of α-syn immunoreactivity in non tg and α-syn tg mice show reduced α-syn levels ipsilateral to the injection sites in the brains of α-syn tg mice that received LV-Atg7 injections. (O–T) Representative sections from the brains of non tg (O, P) and α-syn tg mice (Q–T) that received injections with LV-control (O–R) or LV-Atg7 (S, T) and were immunolabeled with an antibody against MAP2. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (U) Semi-quantitative image analysis of MAP2 immunoreactivity in non tg and α-syn tg mice shows a recovery of MAP2 levels ipsilateral to the injection sites in the brains of α-syn tg mice that received LV-Atg7 injections. Scale bar in panel (F) represents 0.1mm in panels A–F and H–M, 20µm in panels A′–F′ and H′–M′, and 10µm in panels O–T. *p

    Journal: PLoS ONE

    Article Title: Selective Molecular Alterations in the Autophagy Pathway in Patients with Lewy Body Disease and in Models of ?-Synucleinopathy

    doi: 10.1371/journal.pone.0009313

    Figure Lengend Snippet: Immunohistochemical analysis of the effects of LV-Atg7 treatment in α-syn tg mice. For panels A–M, vibratome sections from non tg and α-syn tg mice that received LV injections into the cortex and hippocampus were immunolabeled with antibodies against Atg7 or α-syn and imaged with a digital microscope. Panels A′–M′ represent higher-power images from the hippocampus of the corresponding low-power panels in panels A–M. For panels O–T, effects of rapamycin treatment on α-syn accumulation, autophagy and neuronal integrity in the brains of α-syn tg mice. For panels A–M, vibratome sections from the hippocampus of non tg and α-syn tg mice were immunolabeled with an antibody against MAP2 and imaged with a laser scanning confocal microscope, and images were obtained from the temporal cortex. (A–F) Representative sections from the brains of non tg (A, B) and α-syn tg mice (C–F) that received injections with LV-control (A–D) or LV-Atg7 (E, F) and were immunolabeled with an antibody against Atg7. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (G) Semi-quantitative image analysis of Atg7 immunoreactivity in non tg and α-syn tg mice show increased Atg7 levels ipsilateral to the injection sites in the brains of animals that received LV-Atg7. (H–M) Representative sections from the brains of non tg (H, I) and α-syn tg mice (J–M) that received injections with LV-control (H–K) or LV-Atg7 (L, M) and were immunolabeled with an antibody against α-syn. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (N) Semi-quantitative image analysis of α-syn immunoreactivity in non tg and α-syn tg mice show reduced α-syn levels ipsilateral to the injection sites in the brains of α-syn tg mice that received LV-Atg7 injections. (O–T) Representative sections from the brains of non tg (O, P) and α-syn tg mice (Q–T) that received injections with LV-control (O–R) or LV-Atg7 (S, T) and were immunolabeled with an antibody against MAP2. Images show sections from the hemisphere ipsilateral (ipsi) or contralateral (contra) to the sites of injection. (U) Semi-quantitative image analysis of MAP2 immunoreactivity in non tg and α-syn tg mice shows a recovery of MAP2 levels ipsilateral to the injection sites in the brains of α-syn tg mice that received LV-Atg7 injections. Scale bar in panel (F) represents 0.1mm in panels A–F and H–M, 20µm in panels A′–F′ and H′–M′, and 10µm in panels O–T. *p

    Article Snippet: Additional experiments with the α-syn tg mice included treatments with the autophagy activator rapamycin (Sigma-Aldrich, St. Louis, MO).

    Techniques: Immunohistochemistry, Mouse Assay, Immunolabeling, Microscopy, Injection

    Immunohistochemical and immunoblot analysis of the effects of rapamycin treatment in α-syn tg mice. For panels A–M, vibratome sections from the hippocampus of non tg and α-syn tg mice were immunolabeled with antibodies against α-syn, LC3, Cathepsin D or MAP2 and imaged with a digital microscope. All images are from the temporal cortex. For panel O, brain homogenates from non tg and α-syn tg mice were separated into membrane and lysosomal fractions, and 20 µg of each sample was subjected to gel electrophoresis. (A–C) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against α-syn. (D–F) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against LC3. (G) Semi-quantitative image analysis showing reduced α-syn immunoreactivity and increased LC3 immunoreactivity in the hippocampus of α-syn tg mice treated with Rapamycin. (H–J) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against Cathepsin D. (K–M) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against MAP2. (N) Semi-quantitative image analysis showing increased Cathepsin D immunoreactivity in the hippocampus of α-syn tg mice treated with Rapamycin. Reduced levels of MAP2 in the hippocampus of vehicle-treated α-syn tg mice is rescued by Rapamycin treatment. (O) Representative immunoblot analysis of membrane and lysosomal fractions probed with antibodies against α-syn, LC3, and Cathepsin D. (P) Semi-quantitative image analysis of immunoblots showing redistribution of α-syn from membrane to lysosomal fractions and an associated increase in LC3 and Cathepsin D levels. All semi-quantitative measurements were normalized to actin levels as a loading control. Scale bar in panel (C) represents 40µm in panels A–C, 20µm in panels D–E and H–J, and 10µm in panels K–M. *p

    Journal: PLoS ONE

    Article Title: Selective Molecular Alterations in the Autophagy Pathway in Patients with Lewy Body Disease and in Models of ?-Synucleinopathy

    doi: 10.1371/journal.pone.0009313

    Figure Lengend Snippet: Immunohistochemical and immunoblot analysis of the effects of rapamycin treatment in α-syn tg mice. For panels A–M, vibratome sections from the hippocampus of non tg and α-syn tg mice were immunolabeled with antibodies against α-syn, LC3, Cathepsin D or MAP2 and imaged with a digital microscope. All images are from the temporal cortex. For panel O, brain homogenates from non tg and α-syn tg mice were separated into membrane and lysosomal fractions, and 20 µg of each sample was subjected to gel electrophoresis. (A–C) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against α-syn. (D–F) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against LC3. (G) Semi-quantitative image analysis showing reduced α-syn immunoreactivity and increased LC3 immunoreactivity in the hippocampus of α-syn tg mice treated with Rapamycin. (H–J) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against Cathepsin D. (K–M) Representative sections from the brains of vehicle-treated non tg mice and vehicle- and Rapamycin-treated α-syn tg mice immunolabeled with an antibody against MAP2. (N) Semi-quantitative image analysis showing increased Cathepsin D immunoreactivity in the hippocampus of α-syn tg mice treated with Rapamycin. Reduced levels of MAP2 in the hippocampus of vehicle-treated α-syn tg mice is rescued by Rapamycin treatment. (O) Representative immunoblot analysis of membrane and lysosomal fractions probed with antibodies against α-syn, LC3, and Cathepsin D. (P) Semi-quantitative image analysis of immunoblots showing redistribution of α-syn from membrane to lysosomal fractions and an associated increase in LC3 and Cathepsin D levels. All semi-quantitative measurements were normalized to actin levels as a loading control. Scale bar in panel (C) represents 40µm in panels A–C, 20µm in panels D–E and H–J, and 10µm in panels K–M. *p

    Article Snippet: Additional experiments with the α-syn tg mice included treatments with the autophagy activator rapamycin (Sigma-Aldrich, St. Louis, MO).

    Techniques: Immunohistochemistry, Mouse Assay, Immunolabeling, Microscopy, Nucleic Acid Electrophoresis, Western Blot

    Raptor siRNA mimics the effects of rapa, and mammalian target of rapamycin complex 1 inhibition induces HIF2A expression. (A): SOX9 protein expression levels of chondrogenic pellets cultured in differentiation medium (control) containing 25 nM rapa or

    Journal: Stem Cells Translational Medicine

    Article Title: Rapamycin-Induced Hypoxia Inducible Factor 2A Is Essential for Chondrogenic Differentiation of Amniotic Fluid Stem Cells

    doi: 10.5966/sctm.2015-0262

    Figure Lengend Snippet: Raptor siRNA mimics the effects of rapa, and mammalian target of rapamycin complex 1 inhibition induces HIF2A expression. (A): SOX9 protein expression levels of chondrogenic pellets cultured in differentiation medium (control) containing 25 nM rapa or

    Article Snippet: To selectively block mTOR downstream pathways, 25 nM of the mTORC1 inhibitor rapamycin (Merck Millipore, Billerica, MA, ), 1µM of MK2206, or 1µM of torin 1 (both from Selleckchem, Houston, TX, ) was added to the medium upon preparation and was freshly added when differentiation medium was changed.

    Techniques: Inhibition, Expressing, Cell Culture

    SH-SY5Y cells morphological changes during osteogenic differentiation. SH-SY5Y cells were cultured in osteogenic medium in the presence of hydroxyapatite scaffolds with rapamycin (5 μ M) and analyzed for osteocalcin expression by immunocytochemistry. ( a ) SH-SY5Y cells on the scaffolds during differentiation (in green, osteocalcin; in blue, DAPI; bar=50 μ m). ( b ) Osteocalcin secretion by SH-SY5Y during differentiation (in green, osteocalcin; in blue, DAPI; bar=50 μ m)

    Journal: Cell Death & Disease

    Article Title: Differentiation of human neuroblastoma cells toward the osteogenic lineage by mTOR inhibitor

    doi: 10.1038/cddis.2015.244

    Figure Lengend Snippet: SH-SY5Y cells morphological changes during osteogenic differentiation. SH-SY5Y cells were cultured in osteogenic medium in the presence of hydroxyapatite scaffolds with rapamycin (5 μ M) and analyzed for osteocalcin expression by immunocytochemistry. ( a ) SH-SY5Y cells on the scaffolds during differentiation (in green, osteocalcin; in blue, DAPI; bar=50 μ m). ( b ) Osteocalcin secretion by SH-SY5Y during differentiation (in green, osteocalcin; in blue, DAPI; bar=50 μ m)

    Article Snippet: The differentiation medium was composed as follows: DMEM-F12 with 15 mM HEPES and 2 mM L-glutamine (Lonza, cat. no. BE12-719 F), 10% FBS (Lonza, cat. no. DE14-830 F), 1% penicillin (100 units/ml)/streptomycin (100 mg /ml) (Lonza, cat. no. DE17-602E) and as an osteogenic inductor rapamycin 5 μ M (Calbiochem, Darmstadt, Germany, cat. no. CAS 53123-88-9, Sigma, St. Louis, MI, USA, cat. no. R0395).

    Techniques: Cell Culture, Expressing, Immunocytochemistry

    Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on BACE1 expression in Neuro-2a/amyloid precursor protein 695 cells (western blot assay). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with Rapamycin (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-methyladenine (5 mM). * P

    Journal: Neural Regeneration Research

    Article Title: Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells

    doi: 10.4103/1673-5374.165511

    Figure Lengend Snippet: Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on BACE1 expression in Neuro-2a/amyloid precursor protein 695 cells (western blot assay). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with Rapamycin (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-methyladenine (5 mM). * P

    Article Snippet: Grouping was designed as the following: control group, OGD group (OGD for 1 hour), OGD + Rapamycin group (intervention with 200 ng/mL Rapamycin, Sigma-Aldrich, St. Louis, MO, USA), and OGD + 3-MA group (intervention with 5 mM 3-MA, Sigma-Aldrich).

    Techniques: Expressing, Western Blot

    Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on apoptosis ratio in Neuro-2a/amyloid precursor protein 695 cells (flow cytometry). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with Rapamycin (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-methyladenine (5 mM). In cell clustering of flow cytometer, Q1 represents live cells, Q2 represents necrotic or late apoptotic cells, Q3 represents cells with mechanical injury, and Q4 represents early apoptotic cells. * P

    Journal: Neural Regeneration Research

    Article Title: Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells

    doi: 10.4103/1673-5374.165511

    Figure Lengend Snippet: Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on apoptosis ratio in Neuro-2a/amyloid precursor protein 695 cells (flow cytometry). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with Rapamycin (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-methyladenine (5 mM). In cell clustering of flow cytometer, Q1 represents live cells, Q2 represents necrotic or late apoptotic cells, Q3 represents cells with mechanical injury, and Q4 represents early apoptotic cells. * P

    Article Snippet: Grouping was designed as the following: control group, OGD group (OGD for 1 hour), OGD + Rapamycin group (intervention with 200 ng/mL Rapamycin, Sigma-Aldrich, St. Louis, MO, USA), and OGD + 3-MA group (intervention with 5 mM 3-MA, Sigma-Aldrich).

    Techniques: Flow Cytometry, Cytometry

    Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on microtubule-associated protein 1 light chain 3 (LC3) expression in Neuro-2a/amyloid precursor protein 695 cells (western blot assay). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with Rapamycin (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-MA (5 mM). * P

    Journal: Neural Regeneration Research

    Article Title: Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells

    doi: 10.4103/1673-5374.165511

    Figure Lengend Snippet: Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on microtubule-associated protein 1 light chain 3 (LC3) expression in Neuro-2a/amyloid precursor protein 695 cells (western blot assay). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with Rapamycin (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-MA (5 mM). * P

    Article Snippet: Grouping was designed as the following: control group, OGD group (OGD for 1 hour), OGD + Rapamycin group (intervention with 200 ng/mL Rapamycin, Sigma-Aldrich, St. Louis, MO, USA), and OGD + 3-MA group (intervention with 5 mM 3-MA, Sigma-Aldrich).

    Techniques: Expressing, Western Blot

    Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on Beclin1 expression in Neuro-2a/amyloid precursor protein 695 cells (western blot assay). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with Rapamycin (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-methyladenine (5 mM). * P

    Journal: Neural Regeneration Research

    Article Title: Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells

    doi: 10.4103/1673-5374.165511

    Figure Lengend Snippet: Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on Beclin1 expression in Neuro-2a/amyloid precursor protein 695 cells (western blot assay). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with Rapamycin (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-methyladenine (5 mM). * P

    Article Snippet: Grouping was designed as the following: control group, OGD group (OGD for 1 hour), OGD + Rapamycin group (intervention with 200 ng/mL Rapamycin, Sigma-Aldrich, St. Louis, MO, USA), and OGD + 3-MA group (intervention with 5 mM 3-MA, Sigma-Aldrich).

    Techniques: Expressing, Western Blot

    Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on the morphology and ultrastructure of Neuro-2a/amyloid precursor protein 695 cells (transmission electron microscopy). Control group: Untreated; (B) OGD group: OGD for 1 hour; (C) OGD + Rapa group: pretreated with Rapa (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); (D) OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in presence of 3-methyladenine (5 mM). In B, red arrows indicate autolyosomes. In C, red arrows indicate autophagosomes that are in the process of formation. Blue arrows in every plot mark mitochondria. Scale bars: 500 nm. N: Nucleus; M: mitochondria; AL: autolyosome; AP: autophagosome; 3-MA: 3-methyladenine.

    Journal: Neural Regeneration Research

    Article Title: Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells

    doi: 10.4103/1673-5374.165511

    Figure Lengend Snippet: Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on the morphology and ultrastructure of Neuro-2a/amyloid precursor protein 695 cells (transmission electron microscopy). Control group: Untreated; (B) OGD group: OGD for 1 hour; (C) OGD + Rapa group: pretreated with Rapa (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); (D) OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in presence of 3-methyladenine (5 mM). In B, red arrows indicate autolyosomes. In C, red arrows indicate autophagosomes that are in the process of formation. Blue arrows in every plot mark mitochondria. Scale bars: 500 nm. N: Nucleus; M: mitochondria; AL: autolyosome; AP: autophagosome; 3-MA: 3-methyladenine.

    Article Snippet: Grouping was designed as the following: control group, OGD group (OGD for 1 hour), OGD + Rapamycin group (intervention with 200 ng/mL Rapamycin, Sigma-Aldrich, St. Louis, MO, USA), and OGD + 3-MA group (intervention with 5 mM 3-MA, Sigma-Aldrich).

    Techniques: Transmission Assay, Electron Microscopy

    Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on proliferation rate in Neuro-2a/ amyloid precursor protein 695 cells (cell counting kit-8 assay). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with Rapamycin (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-methyladenine (5 mM). * P

    Journal: Neural Regeneration Research

    Article Title: Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells

    doi: 10.4103/1673-5374.165511

    Figure Lengend Snippet: Effects of autophagy regulation and oxygen-glucose deprivation (OGD) on proliferation rate in Neuro-2a/ amyloid precursor protein 695 cells (cell counting kit-8 assay). Control group: Untreated; OGD group: OGD for 1 hour; OGD + Rapa group: pretreated with Rapamycin (200 ng/mL) for 1 hour followed by OGD for 1 hour in the presence of Rapamycin (200 ng/mL); OGD + 3-MA group: pretreated with 3-methyladenine (5 mM) for 1 hour followed by OGD in the presence of 3-methyladenine (5 mM). * P

    Article Snippet: Grouping was designed as the following: control group, OGD group (OGD for 1 hour), OGD + Rapamycin group (intervention with 200 ng/mL Rapamycin, Sigma-Aldrich, St. Louis, MO, USA), and OGD + 3-MA group (intervention with 5 mM 3-MA, Sigma-Aldrich).

    Techniques: Cell Counting

    In vivo chemical dimerization facilitates microtubule plus end tracking in real time HeLa cells dually transfected with FRB-EGFP and FKBP-EGFP fusions of EB1 and/or CLIP-170. EB1 constructs embody residues 1-185, CLIP-170 constructs residues 3-210 and 129-350. After one minute of imaging, rapamycin was added to the media for a final concentration of 50 nM. Magnified images at right correspond to the boxed region in the first column of the respective row. Images represent a single time point pre-rapamycin treatment and three consecutive time points, taken at 2 sec intervals, post-rapamycin treatment. Arrows, color-coded to the respective time point, track individual microtubule plus ends. Time in sec is indicated relative to rapamycin addition (t = 0 sec). Scale bar in left column, 5 μm.

    Journal: Molecular cell

    Article Title: Structural Basis of Microtubule Plus End Tracking by XMAP215, CLIP-170 and EB1

    doi: 10.1016/j.molcel.2007.07.023

    Figure Lengend Snippet: In vivo chemical dimerization facilitates microtubule plus end tracking in real time HeLa cells dually transfected with FRB-EGFP and FKBP-EGFP fusions of EB1 and/or CLIP-170. EB1 constructs embody residues 1-185, CLIP-170 constructs residues 3-210 and 129-350. After one minute of imaging, rapamycin was added to the media for a final concentration of 50 nM. Magnified images at right correspond to the boxed region in the first column of the respective row. Images represent a single time point pre-rapamycin treatment and three consecutive time points, taken at 2 sec intervals, post-rapamycin treatment. Arrows, color-coded to the respective time point, track individual microtubule plus ends. Time in sec is indicated relative to rapamycin addition (t = 0 sec). Scale bar in left column, 5 μm.

    Article Snippet: For rapamycin-mediated heterodimerization experiments, DMSO or ethanol-solubilized rapamycin (Calbiochem) was added to the media to yield a final concentration of 50 nM.

    Techniques: In Vivo, Transfection, Cross-linking Immunoprecipitation, Construct, Imaging, Concentration Assay, Size-exclusion Chromatography

    Rapamycin rescues laminin α 2 knockdown-mediated changes in the spatial expression of actin binding and regulatory proteins. Sertoli cells cultured for 3 days were transfected with laminin α 2 (Lam α 2) vs negative control (Ctrl) shRNA for 24 hours. Cells were rinsed and treated with rapamycin (100 ng/mL) for 24 hours (for IF). For IB, transfected cells were cultured for an additional 24 hours and were then treated with rapamycin (100 ng/mL) for 24 hours. (a) A study by IB showed that laminin α 2 knockdown and/or rapamycin had no apparent effect on the Arp3 and Eps8 steady-state protein levels. β -actin served as a protein-loading control. (b) A study by IF illustrated that laminin α 2 knockdown caused redistribution of branched actin polymerization protein Arp3 and actin barbed end capping and bundling protein Eps8. These two proteins no longer prominently localized at the cell-cell interface to maintain proper organization of actin filament bundles to support the Sertoli cell TJ barrier function following laminin α 2 knockdown. These proteins were mostly internalized after laminin α 2 knockdown. However, rapamycin blocked laminin α 2 knockdown-induced redistribution of Arp3 and Eps8 in Sertoli cells. GFP expression (green) illustrated successful transfection. Sertoli cell nuclei were visualized by DAPI. Scale bar, 30 μm.

    Journal: Endocrinology

    Article Title: Basement Membrane Laminin α2 Regulation of BTB Dynamics via Its Effects on F-Actin and Microtubule Cytoskeletons Is Mediated Through mTORC1 Signaling

    doi: 10.1210/en.2016-1630

    Figure Lengend Snippet: Rapamycin rescues laminin α 2 knockdown-mediated changes in the spatial expression of actin binding and regulatory proteins. Sertoli cells cultured for 3 days were transfected with laminin α 2 (Lam α 2) vs negative control (Ctrl) shRNA for 24 hours. Cells were rinsed and treated with rapamycin (100 ng/mL) for 24 hours (for IF). For IB, transfected cells were cultured for an additional 24 hours and were then treated with rapamycin (100 ng/mL) for 24 hours. (a) A study by IB showed that laminin α 2 knockdown and/or rapamycin had no apparent effect on the Arp3 and Eps8 steady-state protein levels. β -actin served as a protein-loading control. (b) A study by IF illustrated that laminin α 2 knockdown caused redistribution of branched actin polymerization protein Arp3 and actin barbed end capping and bundling protein Eps8. These two proteins no longer prominently localized at the cell-cell interface to maintain proper organization of actin filament bundles to support the Sertoli cell TJ barrier function following laminin α 2 knockdown. These proteins were mostly internalized after laminin α 2 knockdown. However, rapamycin blocked laminin α 2 knockdown-induced redistribution of Arp3 and Eps8 in Sertoli cells. GFP expression (green) illustrated successful transfection. Sertoli cell nuclei were visualized by DAPI. Scale bar, 30 μm.

    Article Snippet: Rapamycin readymade solution [2.5 mg/mL in dimethyl sulfoxide (DMSO)] was purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Expressing, Binding Assay, Cell Culture, Transfection, Laser Capture Microdissection, Negative Control, shRNA

    Laminin α 2 knockdown by shRNA that causes Sertoli cell MT disorganization is mediated through mTORC1 signaling. Sertoli cells cultured for 3 days were transfected with laminin α 2 (Lam α 2) vs negative control (Ctrl) shRNA for 24 hours. Cells were rinsed and treated with rapamycin (100 ng/mL) for 24 hours before harvested for IF analysis. For IB and MT polymerization assay, transfected cells were cultured for an additional 24 hours and were then treated with rapamycin (100 ng/mL) for 24 hours. (a) Studies by IB illustrated that laminin α 2 knockdown without or with rapamycin treatment had no apparent effect on the expression of MT regulatory proteins EB1, MARK2 and MARK4 vs detyrosinated α -tubulin (the stabilized form of MTs, rendering MTs less dynamic). (b) MT polymerization assay was performed to quantify the ability of cell lysates to induce MT polymerization in Sertoli cells. Laminin α 2 knockdown reduced MT polymerization because considerably less polymerized MTs were detected. However, treatment of Sertoli cells with rapamycin that blocked the function of mTORC1 abolished the laminin α 2 knockdown-induced downregulation of MT polymerization (see pellet). The supernatant contained the monomers of MTs which were similar in all groups. Taxol (paclitaxel) and CaCl 2 served as the corresponding positive and negative controls, which promoted and inhibited MT polymerization, respectively. (c) Studies by IF showed that laminin α 2 knockdown led to MT disorganization as well as mislocalization of EB1 [a +TIP (plus-end tracking protein)] in which EB1 no longer prominently localized with MTs appeared to discrete dots along the MTs. In control cells, α -tubulin, building blocks of MTs, stretched across the entire cell cytosol, whereas MTs retracted from cell cytosol but rounded up to enclose the Sertoli cell nuclei after laminin α 2 knockdown. On the other hand, EB1 associated with long stretches of MTs in control cells, but it no longer prominently found to associate with MTs after laminin α 2 knockdown, likely dispersed in cell cytosol because the EB1 level was not perturbed based on IB [see (a)]. Rapamycin treatment that blocked the mTORC1 signaling rescued laminin α 2 knockdown induced MT disorganization. GFP expression (green) illustrated the successful transfection. Sertoli cell nuclei were visualized by DAPI. Scale bar, 30 μm.

    Journal: Endocrinology

    Article Title: Basement Membrane Laminin α2 Regulation of BTB Dynamics via Its Effects on F-Actin and Microtubule Cytoskeletons Is Mediated Through mTORC1 Signaling

    doi: 10.1210/en.2016-1630

    Figure Lengend Snippet: Laminin α 2 knockdown by shRNA that causes Sertoli cell MT disorganization is mediated through mTORC1 signaling. Sertoli cells cultured for 3 days were transfected with laminin α 2 (Lam α 2) vs negative control (Ctrl) shRNA for 24 hours. Cells were rinsed and treated with rapamycin (100 ng/mL) for 24 hours before harvested for IF analysis. For IB and MT polymerization assay, transfected cells were cultured for an additional 24 hours and were then treated with rapamycin (100 ng/mL) for 24 hours. (a) Studies by IB illustrated that laminin α 2 knockdown without or with rapamycin treatment had no apparent effect on the expression of MT regulatory proteins EB1, MARK2 and MARK4 vs detyrosinated α -tubulin (the stabilized form of MTs, rendering MTs less dynamic). (b) MT polymerization assay was performed to quantify the ability of cell lysates to induce MT polymerization in Sertoli cells. Laminin α 2 knockdown reduced MT polymerization because considerably less polymerized MTs were detected. However, treatment of Sertoli cells with rapamycin that blocked the function of mTORC1 abolished the laminin α 2 knockdown-induced downregulation of MT polymerization (see pellet). The supernatant contained the monomers of MTs which were similar in all groups. Taxol (paclitaxel) and CaCl 2 served as the corresponding positive and negative controls, which promoted and inhibited MT polymerization, respectively. (c) Studies by IF showed that laminin α 2 knockdown led to MT disorganization as well as mislocalization of EB1 [a +TIP (plus-end tracking protein)] in which EB1 no longer prominently localized with MTs appeared to discrete dots along the MTs. In control cells, α -tubulin, building blocks of MTs, stretched across the entire cell cytosol, whereas MTs retracted from cell cytosol but rounded up to enclose the Sertoli cell nuclei after laminin α 2 knockdown. On the other hand, EB1 associated with long stretches of MTs in control cells, but it no longer prominently found to associate with MTs after laminin α 2 knockdown, likely dispersed in cell cytosol because the EB1 level was not perturbed based on IB [see (a)]. Rapamycin treatment that blocked the mTORC1 signaling rescued laminin α 2 knockdown induced MT disorganization. GFP expression (green) illustrated the successful transfection. Sertoli cell nuclei were visualized by DAPI. Scale bar, 30 μm.

    Article Snippet: Rapamycin readymade solution [2.5 mg/mL in dimethyl sulfoxide (DMSO)] was purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: shRNA, Cell Culture, Transfection, Laser Capture Microdissection, Negative Control, Polymerization Assay, Expressing

    Knockdown of laminin α 2 disrupts the Sertoli cell F-actin organization through mTORC1-rpS6-Akt signaling. Primary Sertoli cells cultured for 3 days were transfected with laminin α 2 (Lam α 2) vs nontargeting negative control (Ctrl) shRNA for 24 hours. Cells were rinsed and treated with rapamycin (100 ng/mL) for 24 hours (for IF). For IB, transfected cells were cultured for additional 24 hours and then treated with rapamycin (100 ng/mL) for 24 hours before cells were harvested for lysate preparation. (a) Studies by IF confirmed that transfection of Sertoli cells with laminin α 2 shRNA successfully knockdown laminin α 2, as the expression of laminin α 2 was considerably diminished in the laminin α 2 silenced cells. However, rapamycin blocked the disruptive effect of laminin α 2 knockdown on F-actin organization. Knockdown of laminin α 2 caused truncation and defragmentation of F-actin network, whereas rapamycin rescued this disruptive effect, supporting the notion that laminin α 2 exerts its effects through mTORC1 signaling. GFP expression (green fluorescence) illustrated successful transfection. Sertoli cell nuclei were visualized by DAPI. Scale bar, 30 μm. (b) Studies by IB confirmed that a knockdown of laminin α 2 activated mTORC1-rpS6-Akt signaling by upregulating p -rpS6 S235/236 and p -rpS6. S240/244 and downregulating p -Akt1 S473 and p -Akt2 S474, but not total rpS6 and Akts ( i.e. , Akt1, 2, and 3). Rapamycin inactivated p -rpS6/ p -Akt pathway by abolishing the laminin α 2 knockdown-induced up- and downregulation of p -rpS6 and p -Akt1/2, but rapamycin had no effect on mTOR, raptor, total rpS6, and Akt levels. Actin and α -tubulin served as the protein loading control.

    Journal: Endocrinology

    Article Title: Basement Membrane Laminin α2 Regulation of BTB Dynamics via Its Effects on F-Actin and Microtubule Cytoskeletons Is Mediated Through mTORC1 Signaling

    doi: 10.1210/en.2016-1630

    Figure Lengend Snippet: Knockdown of laminin α 2 disrupts the Sertoli cell F-actin organization through mTORC1-rpS6-Akt signaling. Primary Sertoli cells cultured for 3 days were transfected with laminin α 2 (Lam α 2) vs nontargeting negative control (Ctrl) shRNA for 24 hours. Cells were rinsed and treated with rapamycin (100 ng/mL) for 24 hours (for IF). For IB, transfected cells were cultured for additional 24 hours and then treated with rapamycin (100 ng/mL) for 24 hours before cells were harvested for lysate preparation. (a) Studies by IF confirmed that transfection of Sertoli cells with laminin α 2 shRNA successfully knockdown laminin α 2, as the expression of laminin α 2 was considerably diminished in the laminin α 2 silenced cells. However, rapamycin blocked the disruptive effect of laminin α 2 knockdown on F-actin organization. Knockdown of laminin α 2 caused truncation and defragmentation of F-actin network, whereas rapamycin rescued this disruptive effect, supporting the notion that laminin α 2 exerts its effects through mTORC1 signaling. GFP expression (green fluorescence) illustrated successful transfection. Sertoli cell nuclei were visualized by DAPI. Scale bar, 30 μm. (b) Studies by IB confirmed that a knockdown of laminin α 2 activated mTORC1-rpS6-Akt signaling by upregulating p -rpS6 S235/236 and p -rpS6. S240/244 and downregulating p -Akt1 S473 and p -Akt2 S474, but not total rpS6 and Akts ( i.e. , Akt1, 2, and 3). Rapamycin inactivated p -rpS6/ p -Akt pathway by abolishing the laminin α 2 knockdown-induced up- and downregulation of p -rpS6 and p -Akt1/2, but rapamycin had no effect on mTOR, raptor, total rpS6, and Akt levels. Actin and α -tubulin served as the protein loading control.

    Article Snippet: Rapamycin readymade solution [2.5 mg/mL in dimethyl sulfoxide (DMSO)] was purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Cell Culture, Transfection, Laser Capture Microdissection, Negative Control, shRNA, Expressing, Fluorescence

    Bace2 regulates melanophore dendricity via PI3K/mTOR signaling (A) Scheme for chemical suppressor screen of the bace2 −/− mutant. 24hpf bace2 −/− embryos were treated with each compound from the Sigma LOPAC 1280 library at 30μM for 48 hours, in order to identify chemicals which could rescue the melanophore defects. (B) Compounds were scored with a range of 0 (non-rescued, mutant-like) to 5 (fully rescued, WT-like). (C) Top hits from the screen (with score of 4 and 5) converge on PI3K/mTOR signaling pathway. PI3K inhibitors AS605240 (110nM), Wortmannin (230nM), LY-294,002 (15μM), and mTOR inhibitors Temsirolimus (30μM), PP242 (15μM) all fully rescue the bace2 −/− hyperdendritic melanophores. (D) Quantification of tailfin melanophore cell area at 72hpf with hits from the screen (n=each fish, one-way ANOVA followed by Holm-Sidak’s multiple comparisons test, ****P

    Journal: Developmental cell

    Article Title: Distant insulin signaling regulates vertebrate pigmentation through the sheddase Bace2

    doi: 10.1016/j.devcel.2018.04.025

    Figure Lengend Snippet: Bace2 regulates melanophore dendricity via PI3K/mTOR signaling (A) Scheme for chemical suppressor screen of the bace2 −/− mutant. 24hpf bace2 −/− embryos were treated with each compound from the Sigma LOPAC 1280 library at 30μM for 48 hours, in order to identify chemicals which could rescue the melanophore defects. (B) Compounds were scored with a range of 0 (non-rescued, mutant-like) to 5 (fully rescued, WT-like). (C) Top hits from the screen (with score of 4 and 5) converge on PI3K/mTOR signaling pathway. PI3K inhibitors AS605240 (110nM), Wortmannin (230nM), LY-294,002 (15μM), and mTOR inhibitors Temsirolimus (30μM), PP242 (15μM) all fully rescue the bace2 −/− hyperdendritic melanophores. (D) Quantification of tailfin melanophore cell area at 72hpf with hits from the screen (n=each fish, one-way ANOVA followed by Holm-Sidak’s multiple comparisons test, ****P

    Article Snippet: AS605240: 110nM, 24hpf-72hpf (Sigma, catalog # A0233) Wortmannin: 230nM, 24hpf-72hpf (Sigma, catalog # W1628) LY-294,002: 15μM, 24hpf-72hpf (Sigma, catalog # L9908) Temsirolimus: 30μM, 24hpf-72hpf (Sigma, catalog # PZ0020) PP242: 15μM, 24hpf-72hpf (Abcam, catalog # ab141405) BMS-754807: 7.5μM, 24hpf-72hpf (Sigma, catalog # BM0003) NVP-AEW541: 60μM, 24hpf-72hpf (Selleck Chemicals, catalog # S1034) PTU: 300μM, 3dpF-5dpF (Sigma, catalog # P7629).

    Techniques: Mutagenesis, Fluorescence In Situ Hybridization

    Enhanced Akt-rpS6 activation and in vitro inhibition of rpS6 activation in Oo Pten −/− oocytes by rapamycin. ( A ) Comparison of Akt-rpS6 signaling in Oo Pten −/− and Oo Pten +/+ oocytes. Oocytes were isolated from ovaries of mice at postnatal day 12–14 and immunoblotting was performed as described in Materials and Methods . Loss of PTEN led to enhanced PI3K signaling as indicated by an increase in phosphorylated Akt (p-Akt). The level of phosphorylated rpS6 (p-rpS6) was also increased in Oo Pten −/− oocytes compared with Oo Pten +/+ oocytes. Levels of total rpS6, Akt, and β-actin were used as internal controls. ( B ) Activation of rpS6 in Oo Pten −/− oocytes is dependent on mTORC1 signaling. Oocytes were isolated from ovaries of Oo Pten −/− mice at PD 12–14 as described in Materials and Methods . Treatment of oocytes with the mTORC1-specific inhibitor rapamycin (Rapa, 50 nM) for 2 h was found to largely suppress levels of phosphorylated rpS6 (p-rpS6), but did not affect the level of phosphorylated Akt (p-Akt). As a control, treatment of Oo Pten −/− oocytes with the PI3K-specific inhibitor LY294002 (LY, 50 µM) for 2 h also largely suppressed levels of phosphorylated rpS6 (p-rpS6), but it also suppressed the level of phosphorylated Akt (p-Akt). This suggests that activation of rpS6 in Oo Pten −/− oocytes is dependent on both PI3K and mTORC1 signaling. Levels of total Akt, rpS6, and β-actin were used as internal controls.

    Journal: PLoS ONE

    Article Title: Pharmacological Inhibition of mTORC1 Prevents Over-Activation of the Primordial Follicle Pool in Response to Elevated PI3K Signaling

    doi: 10.1371/journal.pone.0053810

    Figure Lengend Snippet: Enhanced Akt-rpS6 activation and in vitro inhibition of rpS6 activation in Oo Pten −/− oocytes by rapamycin. ( A ) Comparison of Akt-rpS6 signaling in Oo Pten −/− and Oo Pten +/+ oocytes. Oocytes were isolated from ovaries of mice at postnatal day 12–14 and immunoblotting was performed as described in Materials and Methods . Loss of PTEN led to enhanced PI3K signaling as indicated by an increase in phosphorylated Akt (p-Akt). The level of phosphorylated rpS6 (p-rpS6) was also increased in Oo Pten −/− oocytes compared with Oo Pten +/+ oocytes. Levels of total rpS6, Akt, and β-actin were used as internal controls. ( B ) Activation of rpS6 in Oo Pten −/− oocytes is dependent on mTORC1 signaling. Oocytes were isolated from ovaries of Oo Pten −/− mice at PD 12–14 as described in Materials and Methods . Treatment of oocytes with the mTORC1-specific inhibitor rapamycin (Rapa, 50 nM) for 2 h was found to largely suppress levels of phosphorylated rpS6 (p-rpS6), but did not affect the level of phosphorylated Akt (p-Akt). As a control, treatment of Oo Pten −/− oocytes with the PI3K-specific inhibitor LY294002 (LY, 50 µM) for 2 h also largely suppressed levels of phosphorylated rpS6 (p-rpS6), but it also suppressed the level of phosphorylated Akt (p-Akt). This suggests that activation of rpS6 in Oo Pten −/− oocytes is dependent on both PI3K and mTORC1 signaling. Levels of total Akt, rpS6, and β-actin were used as internal controls.

    Article Snippet: Oocytes were incubated with 50 µM of the PI3K-specific inhibitor LY294002 (EMD Biosciences, San Diego, CA) or 50 nM of the mTORC1-specific inhibitor rapamycin (EMD Biosciences, San Diego, CA) for 2 h before being lysed in the lysis buffer.

    Techniques: Activation Assay, In Vitro, Inhibition, Isolation, Mouse Assay