rapamycin Millipore Search Results


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  • 99
    Millipore rapamycin
    Figure 10. <t>Rapamycin</t> (Rap) enhanced high glucose toxic effect on adult mouse cardiomyocytes, whereas 3-methyladenine (3-MA) attenuated it. Cardiomyocytes were isolated from adult C57B/6 mice, cultured under the indicated glucose conditions for
    Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc akt
    Stress- and growth-related proteins and left ventricular assist device (LVAD) support. A−G : total and phosphorylated (p-)ERK42 ( A ), total and p-ERK44 ( B ), total and <t>p-p38</t> MAPK ( C ), total and <t>p-Akt</t> ( D ), total and p-mechanistic target of rapamycin (mTOR; E ), corticotropin-releasing factor 1 (CRF1; F ), and CRF2 ( G ) in normal hearts ( n = 4) and samples from failing hearts before (pre-LVAD; n = 15) and after (post-LVAD; n = 15) LVAD implantation with representative blots. Molecular variables were compared using one-way ANOVA with post hoc Bonferroni analysis. Simple linear regression analysis was used to determine the effect of LVAD time as a continuous variable in clinical and molecular measurements. A t -test was used to compare the effect on LVAD duration as a categorical variable in the change in molecular measurement with LVAD. * P
    Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 53841 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti akt
    miR-150-5p inhibited <t>VEGFA/VEGFR2/Akt/mTOR</t> signaling pathway in CRC. Western blot was used to measure the expression of VEGFA, VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR in transfected HCT116 and HCT8 cells. GAPDH was used as a loading control. Data are shown as the mean±SD of three independent experiments. * p
    Anti Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 17252 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore wortmannin
    HNE impairs lysosomal function. (A and B) Neurons were first treated with 0.05% ethanol (Con) or 100 nM bafilomycin (Baf) for 24 hours, or with 5 µM HNE for 2 and 24 hours, then incubated in the presence of LysoSensor green for 5 minutes and images of LysoSensor fluorescence were acquired. Representative images of LysoSensor fluorescence are shown in panel A and results of quantification of average fluorescence pixel intensity per cell body are shown in panel B (values are the mean and SEM of measurements made on 25 – 35 neurons in 3 separate cultures for each condition. Scale bar in panel A = 100 µm. (C) Neurons plated in black-walled 96-well plates were treated for 6 hours with 0.05% ethanol (Con) 5 µM HNE or 100 nM bafilomycin A (Baf) and then subjected to the DQ-BSA protocol; fluorescence intensities were quantified using a plate reader. (D – I) Neurons were treated for 6 hours with 0.05% ethanol (Con) 5 µM HNE or 100 nM bafilomycin A (Baf) and then enzymatic activities (D and G) and protein levels (E, F, H and I) of cathepsins B and D were measured. Values are the mean and SEM of determinations made in 3 separate experiments. (J and K) Neurons were exposed to the indicated individual or combined treatments for 24 hours and neuronal viability was evaluated by MTS (J) and LDH release (K) assays. HNE, 10 µM; Rap: 100 nM rapamycin; Baf, 100 nM bafilomycin A; WRT, 5 µM <t>wortmannin.</t> Values are the mean and SEM of determinations made in 3 independent experiments. Differences among groups were analyzed by Student’s t-test. *p
    Wortmannin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9936 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc mtor
    Effect of incubation time on the expression levels of autophagy-associated proteins in SCC25 cells treated with 5 μM PLB over 48 hours. Notes: : ( A ) Bar graph shows the expression levels of <t>p-PI3K</t> and PI3K in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( B ) bar graph shows the expression levels of p-GSK3β and GSK3β in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( C ) bar graph shows the expression levels of p-p38 MAPK and p38 MAPK in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( D ) bar graph shows the expression levels of p-Akt and Akt in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( E ) bar graph shows the expression levels of <t>p-mTOR</t> and mTOR in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( F ) bar graph shows the expression levels of LC3-I and LC3-II in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( G ) bar graph shows the expression levels of beclin 1 in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( H ) bar graph shows the ratio of p-pI3K over PI3K in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( I ) bar graph shows the ratio of p-GSK3β over GSK3β in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( J ) bar graph shows the ratio of p-p38 MAPK over p38 MAPK in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( K ) bar graph shows the ratio of p-Akt over Akt in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( L ) bar graph shows the ratio of p-mTOR over mTOR in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; and ( M ) bar graph shows the ratio of LC3-II over LC3-I in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. * P
    Mtor, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 10231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bafilomycin a1
    Pro-inflammatory cytokines stimulate the AMPK-ULK-1 axis while inhibiting mTORC1 in β-cells a–d Prevalence of apoptosis was evaluated by HO-PI staining in INS-1E cells a, c or primary rat islets b, d treated or not (ctrl) for 16 h with IL-1β + IFNγ (cyt), alone or in combination with chloroquine (CQ; 10 µM), <t>Bafilomycin</t> A1 (Baf; 100 nM), 3-Methyladenine (3-MA, 5mM), rapamycin (rap; 100 nM), or torin1 (1–100 nM, as indicated). Data are mean ± SEM of 4–6 independent experiments. * P
    Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 3 methyladenine
    Influence of autophagy stimulator or inhibitors on aurantiamide acetate-induced cell death. U87 cells were incubated with 25 μM aurantiamide acetate (AA), 10 nM rapamycin (Rapa), 10 mM <t>3-methyladenine</t> (3-MA), 40 nM Bafilomycin A1 (BafA1), 10 μM chloroquine (CQ), 25 μM AA plus 10 nM Rapa, 25 μM AA plus 10 mM 3-MA, 25 μM AA plus 40 nM BafA1, or 25 μM AA plus 10 μM CQ. Cells without drug treatment were used as control (Ctrl). (A) Representative images were presented; scale bar, 50 μm. (B) The number of processes per cell was quantified from six micrographs. At least 30 cells per micrograph were analysed. * P
    3 Methyladenine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4065 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho akt
    MAGI1 Knockdown functions through Wnt/β-catenin and <t>PTEN/AKT</t> signaling pathway in LN229 (PTEN wild-type) and glioma stem cells (GSCs). ( A ) The protein expression levels of β-catenin, cyclin D1, PTEN and p-AKT were measured by Western blotting in LN229 and GSCs. ( B ) The ratios of β-catenin, cyclin D1, PTEN and p-AKT were determined by giving a mean net density in LN229 and GSCs. Data were presented as the means ± standard errors. ** p
    Anti Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7466 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt ser473
    FPL64176 upregulates PKCα/βII, ERK1/2 and <t>Akt</t> phosphorylation in brainstem in a LTCC antagonist nifedipine-sensitive manner A–B) Time-courses of FPL64176-induced PKCα/βII, ERK1/2 and Akt phosphorylation the least shrew brainstem. Shrews (n =3 per group) were injected with either vehicle (0 min) or FPL 64176 (10 mg/kg., i.p.). Brainstems were collected at 0, 5, 10, 15, 20, 25, 30 and 60 min. Phospho-PKCα/βII at Thr638/641 (pPKCα/βII), GAPDH, phospho-ERK1/2 at Thr202/204 (pERK1/2), ERK1/2, phospho-Akt at <t>Ser473</t> (pAkt) and Akt of protein samples extracted from individual brainstems were determined by Western blots. Panel A shows representative blots. Panel B shows summarized data. Bands were quantified using ImageJ software. Ratios of pPKCα/βII (~ 80 kD) to GAPDH (~ 37 kD), pERK1/2 (42/44 kD) to ERK1/2 and pAkt (~ 60 kD) to Akt were calculated and expressed as fold change (mean ± S.E.M.) of control (0 min). * P
    Phospho Akt Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8045 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore polysorbate 80
    In vitro  release of plumbagin from oleic acid-based nanoemulsion (polysorbate 80 (3.5%) as a function of time. SGF, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) at 37°C was used as release media.
    Polysorbate 80, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 527 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc phospho mtor
    Changes in expression of antioxidant enzyme, oxidative stress marker, mitochondrial activity, and proteins involved in mitochondrial function after exposure to ozone in IL-1α-pretreated conjunctival epithelial cells. (A) Antioxidant enzyme and oxidative stress marker. (B) Mitochondrial activity and proteins involved in mitochondrial function. IL, interleukin; Mn-SOD, manganese superoxide dismutase; TRXr-1, thioredoxin reductase-1; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; <t>mTOR,</t> mammalian target of <t>rapamycin;</t> p-mTOR, phospho-mammalian target of rapamycin. Error bars represent the standard error of the mean (* p
    Phospho Mtor, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 2472 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Cell Signaling Technology Inc anti mtor
    The <t>mTOR/S6K1</t> signaling pathway is not activated in response to the oxidative stress produced by H 2 O 2 . MCF7 cells were transfected with the indicated siRNAs 72 h before treatment with 0.4 mM H 2 O 2 for 30 min. Cell lysates were analyzed by Western blot with the indicated antibodies. Molecular weight markers are indicated on the left. NT means non-targeting control.
    Anti Mtor, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 3367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore chloroquine
    The <t>mTOR/S6K1</t> signaling pathway is not activated in response to the oxidative stress produced by H 2 O 2 . MCF7 cells were transfected with the indicated siRNAs 72 h before treatment with 0.4 mM H 2 O 2 for 30 min. Cell lysates were analyzed by Western blot with the indicated antibodies. Molecular weight markers are indicated on the left. NT means non-targeting control.
    Chloroquine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pd98059
    Bortezomib (BTZ) induces the phosphorylation of ERK, reduces the level of cathepsin B (CTSB), and suppresses the catalytic process in autophagic lysosomes. ( a ) TOV112D cells were treated with 0.05 μ M BTZ for 24 h. CTSB protein and mRNA levels were measured by immunoblotting and real-time quantitative PCR, respectively. Results shown are mean±standard error for three independent experiments. ( b ) The effects of the overexpression of CTSB for 72 h on the TOV112D cells treated with 0.05 μ M BTZ for 24 h were analyzed by measuring the p62 protein level and ( c ) using the MTT assays. Forced expression of C9-CTSB was detected by anti-C9 antibody with western blot analysis. Results shown are the mean±standard error for three independent experiments. ( d ) The effects induced by the treatment with 0.05 μ M BTZ and 30 μ M <t>PD98059</t> for 24 h were analyzed in TOV112D cells using immunoblotting for the levels of ERK phosphorylation, CTSB, and p62. ( e ) The effect of forced expression of a constitutively active p-ERK (Y204D) for 72 h in TOV112D cells were analyzed by measuring the level of CTSB. Results shown are mean ± standard error for three independent experiments. ( f ) TOV112D cells were transfected with a C9-CTSB expression vector and treated with 0.05 μ M BTZ. After lysosomal labeling with 300 nM Lysotracker (red) for 16 h, the colocalization of CTSB and functional lysosomes was shown by yellow merged signals of anti-C9 (green) into Lysotracker (red). Scale bar represents 30 μ m
    Pd98059, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8744 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ampk inhibitor compound c
    The involvement of <t>AMPK</t> in insulin-induced vasodilation in the presence of PVAT through the secretion of adiponectin. A : The AMPK inhibitor <t>Compound</t> C (Cmpd C) inhibits the insulin-induced vasodilation nonsignificantly in the presence of C57BL/6 PVAT (black square, C57BL/6 resistance artery [RA], with C57BL/6 PVAT [ n = 10]; black circle, C57BL/6 RA in the presence of C57BL/6 PVAT and compound C [ n = 7]). B : PVAT from C57BL/6 mice induces significant threonine 172 phosphorylation of AMPK, as does PVAT from db / db mice. Threonine 172 phosphorylation of AMPK was, however, not significantly different between the two types of PVAT. * P
    Ampk Inhibitor Compound C, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 286 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho akt ser473
    Top : <t>Akt</t> Ser 473 phosphorylation (pAkt <t>Ser473</t> ) in epitrochlearis and soleus muscles with 0, 1.2, or 30 nM insulin. Middle : pAkt1 Ser473 in epitrochlearis and soleus muscles with 0, 1.2, or 30 nM insulin. Muscle lysate was immunoprecipitated with Akt1 antibody
    Anti Phospho Akt Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3394 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cicloheximide
    Regulation of TcADKn nucleolar localization. A) Fluorescence microscopy of T. cruzi epimastigotes in exponential growth phase, treated with Control (control), actinomycin D 10 µg.mL −1 for 4 h, (ActD), puromycin 200 µg.mL −1 for 4 h (Puro), <t>cycloheximide</t> 50 µg.mL −1 for 4 h (CHC), rapamycin 100 µM 12 h (Rapa), hydrogen peroxide 200 µM for 1 h (H 2 O 2 ) and phleomycin 100 µg.mL −1 1 hour (Phleo). Drugs were provided by Sigma. B) For rapamycin treatment, fluorescence was quantified for 40 treated and untreated parasites. In each parasite the fluorescence from the green channel (GFP) was quantified in an area selected according to blue signal (DAPI) fluorescence (nucleus) using the RGB plugin in ImageJ ( http://rsb.info.nih.gov/ij ). Cytoplasmic fluorescence was quantified in the same way selecting the brightest perinuclear areas in the green channel (GFP). Selection criterion was the same for all transfected parasites. Bars represent mean ± S.D. Statistically significant difference was calculated by t-student test (p
    Cicloheximide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mg132
    Regulation of eEF2K in cells expressing BOP1Δ. ( A ) T-REx cells were treated for 15 h with 1 µg/ml doxycycline and then for 1 h with 20 µg of <t>MG132.</t> Cell lysates were subjected to western blot. ( B ) T-REx cells were treated for 15 h with 1 µg/ml doxycycline and treated with PP242 (1 µM, 15 h). Total RNA was extracted and subjected to RT-qPCR analysis for eEF2K and β-actin mRNAs. The significance was determined by t -test. ( C ) T-REx cells expressing BOP1Δ were cultured in complete medium with/without doxycycline. After 33 h, in some cases, cells were treated with 1 µM PP242 or 100 nM rapamycin for 15 h. Cells were lysed and 20 µg of lysate proteins were used for western blot analysis. ( D ) T-REx cells inducibly expressing BOP1Δ were cultured in complete medium with/without doxycycline. After 36 h, in some cases, cells were treated overnight with 10 µM PF-4708671 or 100 nM rapamycin. Cells were lysed and 20 µg of protein were used for western blot analysis or to measure the activity of eEF2K using eEF2 and [γ- 32 P]ATP as substrates.
    Mg132, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dmso
    MCF7 Snail-6SA has increased Erk phosphorylation and decreased sensitivity to higher doses of <t>rapamycin</t> A. Snail, E-cadherin and vimentin expression was assessed in stably transfected MCF7 snail wild-type (Sn-WT) and MCF7 snail mutant (Sn-2SA and Sn-6SA) cell lines by western blotting. B. MCF7 snail wild-type (Snail-WT) and mutant (Snail-6SA) cell lines were treated with <t>DMSO</t> 0.1% or rapamycin 100 nM daily for 3 days. EMT, MAPK and mTOR pathway markers were assessed by western blotting. C. Rapamycin sensitivity in MCF7 snail wild-type (Snail-WT) or mutant (Snail-6SA) cell lines were assessed by SRB assay following 96-hour treatment with increasing doses of rapamycin. ** p
    Dmso, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55328 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sb203580
    Autophagic Activity is Inhibited by Rhein through the MAPKs Signaling Pathways. ( A ) NRK-52E cells were treated with HBSS for 0, 0.5, 1, 2 and 6 hours and subjected to western blot analysis of the phosphorylation of p38 (p-p38), Erk (p-Erk) and JNK (p-JNK). ( B ) NRK-52E cells were exposed to HBSS with or without rhein 5 μg/ml for 6 hours and subjected to western blot analysis of p-p38. ( C ) NRK-52E cells were exposed to HBSS with or without <t>SB203580</t> (a p38 inhibitor) 10 μM for 1 hour and subjected to western blot analysis of LC3 I/II and p-p38. ( D ) NRK-52E cells were exposed to HBSS with or without rhein 5 μg/ml for 6 hours and subjected to western blot analysis of p-Erk. ( E ) NRK-52E cells were exposed to HBSS with or without PD098059 (a p-Erk inhibitor) 50 μM for 1 hour and subjected to western blot analysis of LC3 I/II and p-Erk. ( F ) NRK-52E cells were transfected with mRFP-LC3 and treated with HBSS and bafilomycin A1 10 nM with or without SB203580 10 μM or PD098059 50 μM for 2 hours and subjected to fluorescence microscopy. Scale bar = 5 μm. Data are expressed as mean ± SD, * P
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    Millipore actinomycin d
    Iron reduces TNF, but not IL1β, mRNA stability under hypoxia. ( A and B ) Caco-2 cells were subjected to hypoxia (0.2% O 2 ) for 24 hours in the presence or absence of 150 μmol/L DFO or 200 μmol/L FAC. Cells then were incubated with 5 μg/mL <t>actinomycin</t> D for 1 and 2 hours and the consequent decay of ( A ) TNF and ( B ) IL1β mRNA was monitored by quantitative PCR. Results represent means + SEM of 2 independent experiments performed in triplicate. * P
    Actinomycin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12624 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Figure 10. Rapamycin (Rap) enhanced high glucose toxic effect on adult mouse cardiomyocytes, whereas 3-methyladenine (3-MA) attenuated it. Cardiomyocytes were isolated from adult C57B/6 mice, cultured under the indicated glucose conditions for

    Journal: Autophagy

    Article Title: Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury

    doi: 10.4161/auto.18980

    Figure Lengend Snippet: Figure 10. Rapamycin (Rap) enhanced high glucose toxic effect on adult mouse cardiomyocytes, whereas 3-methyladenine (3-MA) attenuated it. Cardiomyocytes were isolated from adult C57B/6 mice, cultured under the indicated glucose conditions for

    Article Snippet: Rapamycin (Sigma, R0395) was dissolved in absolute ethanol (Fisher Scientific, AC61508), 3-methyladenine (Sigma, M9281) in DMEM (GIBCO, 11966), and Bafilomycin A1 (LC Laboratories, B-1080) in dimethyl sulfoxide (DMSO; Sigma, 472301).

    Techniques: Isolation, Mouse Assay, Cell Culture

    Figure 9. mTORC1 mediated high glucose-induced phosphorylation of ULK1. Cardiomyocytes were cultured under different doses of glucose for 72 h. (A) The phosphorylation of ULK1 at Ser467 was determined by western blot analysis. (B) Rapamycin (Rap)

    Journal: Autophagy

    Article Title: Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury

    doi: 10.4161/auto.18980

    Figure Lengend Snippet: Figure 9. mTORC1 mediated high glucose-induced phosphorylation of ULK1. Cardiomyocytes were cultured under different doses of glucose for 72 h. (A) The phosphorylation of ULK1 at Ser467 was determined by western blot analysis. (B) Rapamycin (Rap)

    Article Snippet: Rapamycin (Sigma, R0395) was dissolved in absolute ethanol (Fisher Scientific, AC61508), 3-methyladenine (Sigma, M9281) in DMEM (GIBCO, 11966), and Bafilomycin A1 (LC Laboratories, B-1080) in dimethyl sulfoxide (DMSO; Sigma, 472301).

    Techniques: Cell Culture, Western Blot

    Figure 3. Rapamycin (Rap) enhanced high-glucose-induced cardiomyocyte death, whereas 3-methyladenine (3-MA) attenuated it. Cardiomyocytes were treated with Rap (50 nM) or 3-MA (2 mM) for 24 h under the indicated glucose conditions. Cell death

    Journal: Autophagy

    Article Title: Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury

    doi: 10.4161/auto.18980

    Figure Lengend Snippet: Figure 3. Rapamycin (Rap) enhanced high-glucose-induced cardiomyocyte death, whereas 3-methyladenine (3-MA) attenuated it. Cardiomyocytes were treated with Rap (50 nM) or 3-MA (2 mM) for 24 h under the indicated glucose conditions. Cell death

    Article Snippet: Rapamycin (Sigma, R0395) was dissolved in absolute ethanol (Fisher Scientific, AC61508), 3-methyladenine (Sigma, M9281) in DMEM (GIBCO, 11966), and Bafilomycin A1 (LC Laboratories, B-1080) in dimethyl sulfoxide (DMSO; Sigma, 472301).

    Techniques:

    Stress- and growth-related proteins and left ventricular assist device (LVAD) support. A−G : total and phosphorylated (p-)ERK42 ( A ), total and p-ERK44 ( B ), total and p-p38 MAPK ( C ), total and p-Akt ( D ), total and p-mechanistic target of rapamycin (mTOR; E ), corticotropin-releasing factor 1 (CRF1; F ), and CRF2 ( G ) in normal hearts ( n = 4) and samples from failing hearts before (pre-LVAD; n = 15) and after (post-LVAD; n = 15) LVAD implantation with representative blots. Molecular variables were compared using one-way ANOVA with post hoc Bonferroni analysis. Simple linear regression analysis was used to determine the effect of LVAD time as a continuous variable in clinical and molecular measurements. A t -test was used to compare the effect on LVAD duration as a categorical variable in the change in molecular measurement with LVAD. * P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Muscle Mechanics and Ventricular Function: Structural and functional cardiac profile after prolonged duration of mechanical unloading: potential implications for myocardial recovery

    doi: 10.1152/ajpheart.00187.2018

    Figure Lengend Snippet: Stress- and growth-related proteins and left ventricular assist device (LVAD) support. A−G : total and phosphorylated (p-)ERK42 ( A ), total and p-ERK44 ( B ), total and p-p38 MAPK ( C ), total and p-Akt ( D ), total and p-mechanistic target of rapamycin (mTOR; E ), corticotropin-releasing factor 1 (CRF1; F ), and CRF2 ( G ) in normal hearts ( n = 4) and samples from failing hearts before (pre-LVAD; n = 15) and after (post-LVAD; n = 15) LVAD implantation with representative blots. Molecular variables were compared using one-way ANOVA with post hoc Bonferroni analysis. Simple linear regression analysis was used to determine the effect of LVAD time as a continuous variable in clinical and molecular measurements. A t -test was used to compare the effect on LVAD duration as a categorical variable in the change in molecular measurement with LVAD. * P

    Article Snippet: The following proteins were analyzed using the indicated antibodies: CaMKII (pan, no. 3362, Cell Signaling, Danvers, MA), Thr286 phosphorylated (p-)CaMKII (no. 3361, Cell Signaling), Thr305 p-CamKII (A0005, One World Laboratory, San Diego, CA), CaM (ab45689, Abcam, Cambridge, MA), MEF2 (ab64644, Abcam), HDAC4 (ab1437, Abcam), HDAC5 (ab1439, Abcam), p-HDAC4 (ab39408, Abcam), p-HDAC5 (sc101692, Santa Cruz Biotechnology, Santa Cruz, CA), MSTN propeptide (MAB7881, R & D Systems, Minneapolis, MN), activin receptor type IIBR (ACTIIBR; ab128544, Abcam), bone morphogenic protein-1 (BMP-1; MAB1927, R & D Systems), SMAD2/3 (no. 3122, Cell Signaling), p-SMAD2 (AB3849, Millipore, Billerica, MA), activin A (ab89307, Abcam), p-p44/42 MAPK (p-ERK1/2; no. 9101, Cell Signaling), p44/42 MAPK (ERK1/2; no. 9102, Cell Signaling), p38 MAPK (no. 9212, Cell Signaling), p-p38 MAPK (no. 9211, Cell Signaling), p-Akt (no. 9271, Cell Signaling), Akt (no. 9272, Cell Signaling), mechanistic target of rapamycin (mTOR; no. 2972, Cell Signaling), p-mTOR (no. 2971, Cell Signaling), corticotropin-releasing factor (CRF) receptor 1 (ab59023, Abcam), CRF receptor 2 (ab104368, Abcam), desmin (D1033, Sigma), smooth muscle myosin heavy chain (MHC; ab124679, Abcam), and matrix metalloproteinase-9 (MMP-9; ab38898, Abcam).

    Techniques:

    Blocking the PI3K/Akt pathway by LY294002 induces apoptosis in MDR breast cancer cells. ( a ) IC 50 value of LY294002 in MCF-7 and MCF-7/A02 (upper panel), Cal51 and CALDOX (lower panel). ( b ) Cells were treated with LY294002 (10 μ M for MCF-7 and MCF-7/A02, 2 μ M for Cal51 and CALDOX) for 48 h. Annexin V/PI staining was detected using flow cytometry. Representative plots of three independent experiments are shown. Quantitative data show the average percentage of annexin V-positive cells (both in early apoptosis, lower right quadrant and late apoptosis, upper right quadrant) of three independent experiments (right panel). ( c ) Caspase-3/7 and caspase-9 activities of MCF-7/A02 (upper histograms) and CALDOX (lower histograms) after LY294002 treatment. ( d ) Fold changes of Bax, Bim and Survivin expression levels determined using RT-qPCR (left panel) and western blot (right panel) in MCF-7/A02 and CALDOX after LY294002 treatment with various concentrations for 48 h. Numerical data are presented as mean±S.D. of three independent replicates. * P

    Journal: Cell Death & Disease

    Article Title: Effects of PI3K inhibitor NVP-BKM120 on overcoming drug resistance and eliminating cancer stem cells in human breast cancer cells

    doi: 10.1038/cddis.2015.363

    Figure Lengend Snippet: Blocking the PI3K/Akt pathway by LY294002 induces apoptosis in MDR breast cancer cells. ( a ) IC 50 value of LY294002 in MCF-7 and MCF-7/A02 (upper panel), Cal51 and CALDOX (lower panel). ( b ) Cells were treated with LY294002 (10 μ M for MCF-7 and MCF-7/A02, 2 μ M for Cal51 and CALDOX) for 48 h. Annexin V/PI staining was detected using flow cytometry. Representative plots of three independent experiments are shown. Quantitative data show the average percentage of annexin V-positive cells (both in early apoptosis, lower right quadrant and late apoptosis, upper right quadrant) of three independent experiments (right panel). ( c ) Caspase-3/7 and caspase-9 activities of MCF-7/A02 (upper histograms) and CALDOX (lower histograms) after LY294002 treatment. ( d ) Fold changes of Bax, Bim and Survivin expression levels determined using RT-qPCR (left panel) and western blot (right panel) in MCF-7/A02 and CALDOX after LY294002 treatment with various concentrations for 48 h. Numerical data are presented as mean±S.D. of three independent replicates. * P

    Article Snippet: The membranes were incubated with primary antibodies to phospho-AKT (D9E), AKT1 (C73H10), Bax (D2E11), Bim (C34C5; Cell Signaling Technology), Survivin (ab76424, Abcam, Cambridge, UK) and NF-κ B p65 (sc372, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C overnight.

    Techniques: Blocking Assay, Staining, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Western Blot

    The PI3K/AKT pathway inhibitor induces apoptosis through suppressing NF- ĸ B activity. ( a ) Western blots show BKM120 downregulating pAKT, nuclear NF- κ B p65 and total NF- κ B p65 in MDR and their parental cells. β -actin was used as a loading control for pAKT, AKT and total NF- κ B p65. Lamin B was used as a loading control for nuclear NF- κ B p65. ( b ) EMSA results show that BKM120 treatments (4 μ M for MCF-7/A02 and 2 μ M for CALDOX) decreased NF- κ B DNA-binding activity in MDR cells. ( c ) Immunofluorescence staining of NF- κ B p65 in MDR cells treated with or without BKM120 (4 μ M for MCF-7/A02 and 2 μ M for CALDOX) for 48 h. ( d ) Western blots show LY294002 treatments (40 μ M for MCF-7/A02 and 4 μ M for CALDOX) downregulating pAKT, nuclear NF- κ B p65 and total NF- κ B p65 in MDR cells. ( e ) MCF-7/A02 and CALDOX cells were transiently transfected with NF- κ B p65 siRNA (siNF- κ B p65) or scrambled siRNA (siControl). Three days after transfection, cells were stained with Annexin V/PI and cell death was quantified using flow cytometry. Representative plots of three independent experiments are shown. Quantitative data show the average percentage of annexin V-positive cells (both in early apoptosis, lower right quadrant, and late apoptosis, upper right quadrant) of three independent experiments (lower panel). ( f ) Fold changes of NF- κ B p65, Bax, Bim and Survivin mRNA levels detected using RT-qPCR in MDR cells after NF- κ B p65 siRNA transfection for 72 h. Numerical data are presented as mean±S.D. of three independent replicates. * P

    Journal: Cell Death & Disease

    Article Title: Effects of PI3K inhibitor NVP-BKM120 on overcoming drug resistance and eliminating cancer stem cells in human breast cancer cells

    doi: 10.1038/cddis.2015.363

    Figure Lengend Snippet: The PI3K/AKT pathway inhibitor induces apoptosis through suppressing NF- ĸ B activity. ( a ) Western blots show BKM120 downregulating pAKT, nuclear NF- κ B p65 and total NF- κ B p65 in MDR and their parental cells. β -actin was used as a loading control for pAKT, AKT and total NF- κ B p65. Lamin B was used as a loading control for nuclear NF- κ B p65. ( b ) EMSA results show that BKM120 treatments (4 μ M for MCF-7/A02 and 2 μ M for CALDOX) decreased NF- κ B DNA-binding activity in MDR cells. ( c ) Immunofluorescence staining of NF- κ B p65 in MDR cells treated with or without BKM120 (4 μ M for MCF-7/A02 and 2 μ M for CALDOX) for 48 h. ( d ) Western blots show LY294002 treatments (40 μ M for MCF-7/A02 and 4 μ M for CALDOX) downregulating pAKT, nuclear NF- κ B p65 and total NF- κ B p65 in MDR cells. ( e ) MCF-7/A02 and CALDOX cells were transiently transfected with NF- κ B p65 siRNA (siNF- κ B p65) or scrambled siRNA (siControl). Three days after transfection, cells were stained with Annexin V/PI and cell death was quantified using flow cytometry. Representative plots of three independent experiments are shown. Quantitative data show the average percentage of annexin V-positive cells (both in early apoptosis, lower right quadrant, and late apoptosis, upper right quadrant) of three independent experiments (lower panel). ( f ) Fold changes of NF- κ B p65, Bax, Bim and Survivin mRNA levels detected using RT-qPCR in MDR cells after NF- κ B p65 siRNA transfection for 72 h. Numerical data are presented as mean±S.D. of three independent replicates. * P

    Article Snippet: The membranes were incubated with primary antibodies to phospho-AKT (D9E), AKT1 (C73H10), Bax (D2E11), Bim (C34C5; Cell Signaling Technology), Survivin (ab76424, Abcam, Cambridge, UK) and NF-κ B p65 (sc372, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C overnight.

    Techniques: Activity Assay, Western Blot, Binding Assay, Immunofluorescence, Staining, Transfection, Flow Cytometry, Cytometry, Quantitative RT-PCR

    The antitumor activity of BKM120 in MCF-7/A02 and CALDOX xenograft tumors. ( a and b ) Tumor sizes of MCF-7/A02 ( a ) and CALDOX ( b ) xenografts after treatment with PBS (control), DOX, BKM120 or BKM120 plus DOX. Data are presented as the mean tumor size±S.D. of six mice per group. ( c and d ) Body weight of nude mice bearing MCF-7/A02 ( c ) and CALDOX ( d ) xenografts treated with PBS (control), DOX, BKM120 or BKM120 plus DOX. Data are presented as the mean body weight±S.D. of six mice per group. ( e ) Western blot analysis of pAKT, AKT, nuclear NF- κ B p65 and total NF- κ B p65 on MCF-7/A02 and CALDOX derived tumors treated with BKM120 or PBS. Tumors were obtained from two mice randomly chosen from six mice per group. Both lamin B and β -actin were used as loading controls. ( f ) Relative fold of Bax, Bim and Survivin gene expression levels in MCF-7/A02 and CALDOX derived tumors treated with BKM120 or PBS. * P

    Journal: Cell Death & Disease

    Article Title: Effects of PI3K inhibitor NVP-BKM120 on overcoming drug resistance and eliminating cancer stem cells in human breast cancer cells

    doi: 10.1038/cddis.2015.363

    Figure Lengend Snippet: The antitumor activity of BKM120 in MCF-7/A02 and CALDOX xenograft tumors. ( a and b ) Tumor sizes of MCF-7/A02 ( a ) and CALDOX ( b ) xenografts after treatment with PBS (control), DOX, BKM120 or BKM120 plus DOX. Data are presented as the mean tumor size±S.D. of six mice per group. ( c and d ) Body weight of nude mice bearing MCF-7/A02 ( c ) and CALDOX ( d ) xenografts treated with PBS (control), DOX, BKM120 or BKM120 plus DOX. Data are presented as the mean body weight±S.D. of six mice per group. ( e ) Western blot analysis of pAKT, AKT, nuclear NF- κ B p65 and total NF- κ B p65 on MCF-7/A02 and CALDOX derived tumors treated with BKM120 or PBS. Tumors were obtained from two mice randomly chosen from six mice per group. Both lamin B and β -actin were used as loading controls. ( f ) Relative fold of Bax, Bim and Survivin gene expression levels in MCF-7/A02 and CALDOX derived tumors treated with BKM120 or PBS. * P

    Article Snippet: The membranes were incubated with primary antibodies to phospho-AKT (D9E), AKT1 (C73H10), Bax (D2E11), Bim (C34C5; Cell Signaling Technology), Survivin (ab76424, Abcam, Cambridge, UK) and NF-κ B p65 (sc372, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C overnight.

    Techniques: Activity Assay, Mouse Assay, Western Blot, Derivative Assay, Expressing

    Schematic diagram of the proposed mechanism underlying HSYA-SDT-induced autophagy and inflammation inhibition in THP-1 macrophages. HSYA-mediated SDT induces an autophagic response through the PI3K/Akt/mTOR signaling pathway mediated by ROS, thereby inhibiting inflammation.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: ROS-Dependent Activation of Autophagy through the PI3K/Akt/mTOR Pathway Is Induced by Hydroxysafflor Yellow A-Sonodynamic Therapy in THP-1 Macrophages

    doi: 10.1155/2017/8519169

    Figure Lengend Snippet: Schematic diagram of the proposed mechanism underlying HSYA-SDT-induced autophagy and inflammation inhibition in THP-1 macrophages. HSYA-mediated SDT induces an autophagic response through the PI3K/Akt/mTOR signaling pathway mediated by ROS, thereby inhibiting inflammation.

    Article Snippet: After the membranes were blocked at room temperature for 1 h in blocking buffer containing 5% dried skim milk diluted with Tris-buffered saline-Tween 20 (TBST), they were incubated with primary antibodies against LC3B (Sigma-Aldrich Co., St. Louis, MO, USA), p62, mTOR, p-mTOR, AKT, p-AKT, Atg5 (Cell Signaling Technology, Inc., USA), beclin 1, IL-1β , IL-12, TNF-α (Abcam, Burlingame, CA, USA), and β -actin (ZSGB-BIO, Inc., Beijing, China, all primary antibodies above were diluted 1 : 1000) at 4°C overnight.

    Techniques: Inhibition

    HSYA-SDT induced autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway in THP-1 macrophages. (a) mTOR, p-mTOR (Ser 2448), AKT, and p-AKT (Ser 473) protein expression was analyzed by Western blots at different time points after HSYA-SDT, and quantifications of the p-mTOR/mTOR ratio and p-AKT/AKT ratio are shown. (b) The effect of LY294002 on the expression levels of mTOR, p-mTOR (Ser 2448), AKT, p-AKT (Ser 473), LC3-Ι, LC3-ΙΙ, p62, and beclin 1 protein at 30 min after SDT, and quantifications of the proteins above are shown. (c) The effect of triciribine on the expression levels of mTOR, p-mTOR (Ser 2448), AKT, p-AKT (Ser 473), LC3-Ι, LC3-ΙΙ, p62, and beclin 1 protein at 30 min after SDT, and quantifications of the proteins above are shown. (d) The effect of rapamycin on the expression levels of mTOR, p-mTOR (Ser 2448), LC3-Ι, LC3-ΙΙ, p62, and beclin 1 protein at 30 min after SDT, and quantifications of the proteins above were shown. (e) The effect of IGF-1 on the expression levels of mTOR, p-mTOR (Ser 2448), AKT, p-AKT (Ser 473), LC3-Ι, LC3-ΙΙ, p62, and beclin 1 protein at 30 min after SDT, and quantifications of the proteins above were shown. All data are mean ± standard error ( n = 5). ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: ROS-Dependent Activation of Autophagy through the PI3K/Akt/mTOR Pathway Is Induced by Hydroxysafflor Yellow A-Sonodynamic Therapy in THP-1 Macrophages

    doi: 10.1155/2017/8519169

    Figure Lengend Snippet: HSYA-SDT induced autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway in THP-1 macrophages. (a) mTOR, p-mTOR (Ser 2448), AKT, and p-AKT (Ser 473) protein expression was analyzed by Western blots at different time points after HSYA-SDT, and quantifications of the p-mTOR/mTOR ratio and p-AKT/AKT ratio are shown. (b) The effect of LY294002 on the expression levels of mTOR, p-mTOR (Ser 2448), AKT, p-AKT (Ser 473), LC3-Ι, LC3-ΙΙ, p62, and beclin 1 protein at 30 min after SDT, and quantifications of the proteins above are shown. (c) The effect of triciribine on the expression levels of mTOR, p-mTOR (Ser 2448), AKT, p-AKT (Ser 473), LC3-Ι, LC3-ΙΙ, p62, and beclin 1 protein at 30 min after SDT, and quantifications of the proteins above are shown. (d) The effect of rapamycin on the expression levels of mTOR, p-mTOR (Ser 2448), LC3-Ι, LC3-ΙΙ, p62, and beclin 1 protein at 30 min after SDT, and quantifications of the proteins above were shown. (e) The effect of IGF-1 on the expression levels of mTOR, p-mTOR (Ser 2448), AKT, p-AKT (Ser 473), LC3-Ι, LC3-ΙΙ, p62, and beclin 1 protein at 30 min after SDT, and quantifications of the proteins above were shown. All data are mean ± standard error ( n = 5). ∗ p

    Article Snippet: After the membranes were blocked at room temperature for 1 h in blocking buffer containing 5% dried skim milk diluted with Tris-buffered saline-Tween 20 (TBST), they were incubated with primary antibodies against LC3B (Sigma-Aldrich Co., St. Louis, MO, USA), p62, mTOR, p-mTOR, AKT, p-AKT, Atg5 (Cell Signaling Technology, Inc., USA), beclin 1, IL-1β , IL-12, TNF-α (Abcam, Burlingame, CA, USA), and β -actin (ZSGB-BIO, Inc., Beijing, China, all primary antibodies above were diluted 1 : 1000) at 4°C overnight.

    Techniques: Expressing, Western Blot

    Autophagy triggered by HSYA-SDT through the PI3K/Akt/mTOR pathway and the inhibition of inflammatory factors were suppressed by the ROS scavenger NAC. (a) The relative fluorescence intensity of ROS generation detected in THP-1 macrophages with or without pretreatment with NAC was measured by flow cytometry. (b) The effect of NAC on the expression levels of mTOR, p-mTOR (Ser 2448), AKT, p-AKT (Ser 473), LC3-Ι, LC3-ΙΙ, p62, and beclin 1 at 30 min after SDT was determined, and quantifications of the proteins above are shown. (c) Protein extracts from untreated cells (Control) and cells treated with NAC, HSYA-SDT, or NAC prior to HSYA-SDT were analyzed by Western blots to detect TNF- α , IL-12, and IL-1 β . Quantifications of protein expression are also provided. (d) ELISAs of the inflammatory factors TNF- α , IL-12, and IL-1 β secreted by THP-1 macrophages with or without pretreatment by NAC. All data are mean ± standard error ( n = 5). ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: ROS-Dependent Activation of Autophagy through the PI3K/Akt/mTOR Pathway Is Induced by Hydroxysafflor Yellow A-Sonodynamic Therapy in THP-1 Macrophages

    doi: 10.1155/2017/8519169

    Figure Lengend Snippet: Autophagy triggered by HSYA-SDT through the PI3K/Akt/mTOR pathway and the inhibition of inflammatory factors were suppressed by the ROS scavenger NAC. (a) The relative fluorescence intensity of ROS generation detected in THP-1 macrophages with or without pretreatment with NAC was measured by flow cytometry. (b) The effect of NAC on the expression levels of mTOR, p-mTOR (Ser 2448), AKT, p-AKT (Ser 473), LC3-Ι, LC3-ΙΙ, p62, and beclin 1 at 30 min after SDT was determined, and quantifications of the proteins above are shown. (c) Protein extracts from untreated cells (Control) and cells treated with NAC, HSYA-SDT, or NAC prior to HSYA-SDT were analyzed by Western blots to detect TNF- α , IL-12, and IL-1 β . Quantifications of protein expression are also provided. (d) ELISAs of the inflammatory factors TNF- α , IL-12, and IL-1 β secreted by THP-1 macrophages with or without pretreatment by NAC. All data are mean ± standard error ( n = 5). ∗ p

    Article Snippet: After the membranes were blocked at room temperature for 1 h in blocking buffer containing 5% dried skim milk diluted with Tris-buffered saline-Tween 20 (TBST), they were incubated with primary antibodies against LC3B (Sigma-Aldrich Co., St. Louis, MO, USA), p62, mTOR, p-mTOR, AKT, p-AKT, Atg5 (Cell Signaling Technology, Inc., USA), beclin 1, IL-1β , IL-12, TNF-α (Abcam, Burlingame, CA, USA), and β -actin (ZSGB-BIO, Inc., Beijing, China, all primary antibodies above were diluted 1 : 1000) at 4°C overnight.

    Techniques: Inhibition, Fluorescence, Flow Cytometry, Cytometry, Expressing, Western Blot

    miR-150-5p inhibited VEGFA/VEGFR2/Akt/mTOR signaling pathway in CRC. Western blot was used to measure the expression of VEGFA, VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR in transfected HCT116 and HCT8 cells. GAPDH was used as a loading control. Data are shown as the mean±SD of three independent experiments. * p

    Journal: Aging (Albany NY)

    Article Title: miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer

    doi: 10.18632/aging.101656

    Figure Lengend Snippet: miR-150-5p inhibited VEGFA/VEGFR2/Akt/mTOR signaling pathway in CRC. Western blot was used to measure the expression of VEGFA, VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR in transfected HCT116 and HCT8 cells. GAPDH was used as a loading control. Data are shown as the mean±SD of three independent experiments. * p

    Article Snippet: The membranes were blocked with 5% bovine serum albumin (BSA) for 1.5 h and then incubated with primary antibodies: rabbit polyclonal anti-VEGFA (1:1000, ab46154, abcam, UK), anti-VEGFR2 (1:1000, ab39256, abcam, UK), anti-phospho(Y1175)-VEGFR2 (1:1000, ab194806, abcam, UK), anti-Akt (#9272, Cell Signaling Technology, USA), anti-phospho (Ser473)-Akt (1:2000, #4060, Cell Signaling Technology, USA), anti-mTOR (1:1000, ab2732, abcam, UK), anti-phospho(S2448)-mTOR (1:1000, ab84400 abcam, UK) and rabbit anti-GAPDH (1:10000, ab9485, abcam, UK).

    Techniques: Western Blot, Expressing, Transfection

    HNE impairs lysosomal function. (A and B) Neurons were first treated with 0.05% ethanol (Con) or 100 nM bafilomycin (Baf) for 24 hours, or with 5 µM HNE for 2 and 24 hours, then incubated in the presence of LysoSensor green for 5 minutes and images of LysoSensor fluorescence were acquired. Representative images of LysoSensor fluorescence are shown in panel A and results of quantification of average fluorescence pixel intensity per cell body are shown in panel B (values are the mean and SEM of measurements made on 25 – 35 neurons in 3 separate cultures for each condition. Scale bar in panel A = 100 µm. (C) Neurons plated in black-walled 96-well plates were treated for 6 hours with 0.05% ethanol (Con) 5 µM HNE or 100 nM bafilomycin A (Baf) and then subjected to the DQ-BSA protocol; fluorescence intensities were quantified using a plate reader. (D – I) Neurons were treated for 6 hours with 0.05% ethanol (Con) 5 µM HNE or 100 nM bafilomycin A (Baf) and then enzymatic activities (D and G) and protein levels (E, F, H and I) of cathepsins B and D were measured. Values are the mean and SEM of determinations made in 3 separate experiments. (J and K) Neurons were exposed to the indicated individual or combined treatments for 24 hours and neuronal viability was evaluated by MTS (J) and LDH release (K) assays. HNE, 10 µM; Rap: 100 nM rapamycin; Baf, 100 nM bafilomycin A; WRT, 5 µM wortmannin. Values are the mean and SEM of determinations made in 3 independent experiments. Differences among groups were analyzed by Student’s t-test. *p

    Journal: Journal of neurochemistry

    Article Title: Early Involvement of Lysosome Dysfunction in the Degeneration of Cerebral Cortical Neurons Caused by the Lipid Peroxidation Product 4-Hydroxynonenal

    doi: 10.1111/jnc.13957

    Figure Lengend Snippet: HNE impairs lysosomal function. (A and B) Neurons were first treated with 0.05% ethanol (Con) or 100 nM bafilomycin (Baf) for 24 hours, or with 5 µM HNE for 2 and 24 hours, then incubated in the presence of LysoSensor green for 5 minutes and images of LysoSensor fluorescence were acquired. Representative images of LysoSensor fluorescence are shown in panel A and results of quantification of average fluorescence pixel intensity per cell body are shown in panel B (values are the mean and SEM of measurements made on 25 – 35 neurons in 3 separate cultures for each condition. Scale bar in panel A = 100 µm. (C) Neurons plated in black-walled 96-well plates were treated for 6 hours with 0.05% ethanol (Con) 5 µM HNE or 100 nM bafilomycin A (Baf) and then subjected to the DQ-BSA protocol; fluorescence intensities were quantified using a plate reader. (D – I) Neurons were treated for 6 hours with 0.05% ethanol (Con) 5 µM HNE or 100 nM bafilomycin A (Baf) and then enzymatic activities (D and G) and protein levels (E, F, H and I) of cathepsins B and D were measured. Values are the mean and SEM of determinations made in 3 separate experiments. (J and K) Neurons were exposed to the indicated individual or combined treatments for 24 hours and neuronal viability was evaluated by MTS (J) and LDH release (K) assays. HNE, 10 µM; Rap: 100 nM rapamycin; Baf, 100 nM bafilomycin A; WRT, 5 µM wortmannin. Values are the mean and SEM of determinations made in 3 independent experiments. Differences among groups were analyzed by Student’s t-test. *p

    Article Snippet: Rapamycin and wortmannin were purchased from Sigma Chemical Company (St. Louis, MO).

    Techniques: Incubation, Fluorescence

    Effect of incubation time on the expression levels of autophagy-associated proteins in SCC25 cells treated with 5 μM PLB over 48 hours. Notes: : ( A ) Bar graph shows the expression levels of p-PI3K and PI3K in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( B ) bar graph shows the expression levels of p-GSK3β and GSK3β in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( C ) bar graph shows the expression levels of p-p38 MAPK and p38 MAPK in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( D ) bar graph shows the expression levels of p-Akt and Akt in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( E ) bar graph shows the expression levels of p-mTOR and mTOR in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( F ) bar graph shows the expression levels of LC3-I and LC3-II in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( G ) bar graph shows the expression levels of beclin 1 in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( H ) bar graph shows the ratio of p-pI3K over PI3K in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( I ) bar graph shows the ratio of p-GSK3β over GSK3β in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( J ) bar graph shows the ratio of p-p38 MAPK over p38 MAPK in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( K ) bar graph shows the ratio of p-Akt over Akt in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( L ) bar graph shows the ratio of p-mTOR over mTOR in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; and ( M ) bar graph shows the ratio of LC3-II over LC3-I in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. * P

    Journal: Drug Design, Development and Therapy

    Article Title: Plumbagin induces G2/M arrest, apoptosis, and autophagy via p38 MAPK- and PI3K/Akt/mTOR-mediated pathways in human tongue squamous cell carcinoma cells

    doi: 10.2147/DDDT.S76057

    Figure Lengend Snippet: Effect of incubation time on the expression levels of autophagy-associated proteins in SCC25 cells treated with 5 μM PLB over 48 hours. Notes: : ( A ) Bar graph shows the expression levels of p-PI3K and PI3K in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( B ) bar graph shows the expression levels of p-GSK3β and GSK3β in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( C ) bar graph shows the expression levels of p-p38 MAPK and p38 MAPK in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( D ) bar graph shows the expression levels of p-Akt and Akt in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( E ) bar graph shows the expression levels of p-mTOR and mTOR in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( F ) bar graph shows the expression levels of LC3-I and LC3-II in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( G ) bar graph shows the expression levels of beclin 1 in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( H ) bar graph shows the ratio of p-pI3K over PI3K in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( I ) bar graph shows the ratio of p-GSK3β over GSK3β in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( J ) bar graph shows the ratio of p-p38 MAPK over p38 MAPK in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( K ) bar graph shows the ratio of p-Akt over Akt in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; ( L ) bar graph shows the ratio of p-mTOR over mTOR in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours; and ( M ) bar graph shows the ratio of LC3-II over LC3-I in SCC25 cells treated with PLB at 5 μM for 6, 24 and 48 hours. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. * P

    Article Snippet: Primary antibodies against human cell division cycle protein 2 homolog (Cdc2), cyclin B1, p53, p21/Waf1, p27 Kip1, Bcl-2-like protein 4/Bcl-2-associated X protein (Bax), Bcl-2, B-cell lymphoma-extra large (Bcl-xl), the p53 upregulated modulator of apoptosis (PUMA), cytochrome c, cleaved caspase 9, cleaved caspase 3, phospho-(p-)PI3K (Tyr458), PI3K, p-p38 (Thr180/Tyr182), p38, p-Akt (Ser473), Akt, p-mTOR (Ser2448), mTOR, beclin 1, microtubule-associated protein 1A/1B-light chain 3 (LC3-I), and LC3-II were all purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

    Techniques: Incubation, Expressing

    Effect of PLB on the expression level of autophagy-associated proteins in SCC25 cells. Notes: The phosphorylation levels of PI3K, GSK3β, p38 MAPK, and Akt, and the total levels of mTOR, beclin 1, LC3-I, and LC3-II in SCC25 cells determined by Western blotting assay. β-actin was used as the internal control. ( A ) Representative blots show the expression levels of p-PI3K, PI3K, p-GSK3β, GSK3β, p-p38 MAPK, p38 MAPK, p-Akt, Akt, p-mTOR, mTOR, beclin 1, LC3-I, and LC3-II in SCC25 cells treated with PLB at 0.1, 1, and 5 μM for 24 hours and ( B ) representative blots show the expression levels of p-PI3K, PI3K, p-GSK3β, GSK3β, p-p38 MAPK, p38 MAPK , p-Akt, Akt, p-mTOR, mTOR, beclin 1, LC3-I, and LC3-II in SCC25 cells treated with PLB at 5 μM for 6, 24, and 48 hours. β-actin was used as the internal control. Abbreviation: Akt, protein kinase B; GSK3β, glycogen synthase kinase 3β; LC3, microtubule-associated protein 1 light chain 3; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositide 3 kinase; PLB, plumbagin.

    Journal: Drug Design, Development and Therapy

    Article Title: Plumbagin induces G2/M arrest, apoptosis, and autophagy via p38 MAPK- and PI3K/Akt/mTOR-mediated pathways in human tongue squamous cell carcinoma cells

    doi: 10.2147/DDDT.S76057

    Figure Lengend Snippet: Effect of PLB on the expression level of autophagy-associated proteins in SCC25 cells. Notes: The phosphorylation levels of PI3K, GSK3β, p38 MAPK, and Akt, and the total levels of mTOR, beclin 1, LC3-I, and LC3-II in SCC25 cells determined by Western blotting assay. β-actin was used as the internal control. ( A ) Representative blots show the expression levels of p-PI3K, PI3K, p-GSK3β, GSK3β, p-p38 MAPK, p38 MAPK, p-Akt, Akt, p-mTOR, mTOR, beclin 1, LC3-I, and LC3-II in SCC25 cells treated with PLB at 0.1, 1, and 5 μM for 24 hours and ( B ) representative blots show the expression levels of p-PI3K, PI3K, p-GSK3β, GSK3β, p-p38 MAPK, p38 MAPK , p-Akt, Akt, p-mTOR, mTOR, beclin 1, LC3-I, and LC3-II in SCC25 cells treated with PLB at 5 μM for 6, 24, and 48 hours. β-actin was used as the internal control. Abbreviation: Akt, protein kinase B; GSK3β, glycogen synthase kinase 3β; LC3, microtubule-associated protein 1 light chain 3; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositide 3 kinase; PLB, plumbagin.

    Article Snippet: Primary antibodies against human cell division cycle protein 2 homolog (Cdc2), cyclin B1, p53, p21/Waf1, p27 Kip1, Bcl-2-like protein 4/Bcl-2-associated X protein (Bax), Bcl-2, B-cell lymphoma-extra large (Bcl-xl), the p53 upregulated modulator of apoptosis (PUMA), cytochrome c, cleaved caspase 9, cleaved caspase 3, phospho-(p-)PI3K (Tyr458), PI3K, p-p38 (Thr180/Tyr182), p38, p-Akt (Ser473), Akt, p-mTOR (Ser2448), mTOR, beclin 1, microtubule-associated protein 1A/1B-light chain 3 (LC3-I), and LC3-II were all purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

    Techniques: Expressing, Western Blot

    Effect of PLB concentration on the expression levels of autophagy-associated proteins in SCC25 cells. Notes: : ( A ) Bar graph shows the expression levels of p-PI3K and PI3K in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( B ) bar graph shows the expression levels of p-GSK3β and GSK3β in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( C ) bar graph shows the expression levels of p-p38 MAPK and p38 MAPK in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( D ) bar graph shows the expression levels of p-Akt and Akt in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( E ) bar graph shows the expression levels of p-mTOR and mTOR in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( F ) bar graph shows the expression levels of LC3-I and LC3-II in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( G ) bar graph shows the expression levels of beclin 1 in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( H ) bar graph shows the ratio of p-pI3K over PI3K in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( I ) bar graph shows the ratio of p-GSK3β over GSK3β in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( J ) bar graph shows the ratio of p-p38 MAPK over p38 MAPK in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( K ) bar graph shows the ratio of p-Akt over Akt in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( L ) bar graph shows the ratio of p-mTOR over mTOR in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; and ( M ) bar graph shows the ratio of LC3-II over LC3-I in SCC25 cells treated with PLB at 0.1, 1, and 5 μM. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. * P

    Journal: Drug Design, Development and Therapy

    Article Title: Plumbagin induces G2/M arrest, apoptosis, and autophagy via p38 MAPK- and PI3K/Akt/mTOR-mediated pathways in human tongue squamous cell carcinoma cells

    doi: 10.2147/DDDT.S76057

    Figure Lengend Snippet: Effect of PLB concentration on the expression levels of autophagy-associated proteins in SCC25 cells. Notes: : ( A ) Bar graph shows the expression levels of p-PI3K and PI3K in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( B ) bar graph shows the expression levels of p-GSK3β and GSK3β in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( C ) bar graph shows the expression levels of p-p38 MAPK and p38 MAPK in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( D ) bar graph shows the expression levels of p-Akt and Akt in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( E ) bar graph shows the expression levels of p-mTOR and mTOR in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( F ) bar graph shows the expression levels of LC3-I and LC3-II in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( G ) bar graph shows the expression levels of beclin 1 in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( H ) bar graph shows the ratio of p-pI3K over PI3K in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( I ) bar graph shows the ratio of p-GSK3β over GSK3β in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( J ) bar graph shows the ratio of p-p38 MAPK over p38 MAPK in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( K ) bar graph shows the ratio of p-Akt over Akt in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; ( L ) bar graph shows the ratio of p-mTOR over mTOR in SCC25 cells treated with PLB at 0.1, 1, and 5 μM; and ( M ) bar graph shows the ratio of LC3-II over LC3-I in SCC25 cells treated with PLB at 0.1, 1, and 5 μM. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. * P

    Article Snippet: Primary antibodies against human cell division cycle protein 2 homolog (Cdc2), cyclin B1, p53, p21/Waf1, p27 Kip1, Bcl-2-like protein 4/Bcl-2-associated X protein (Bax), Bcl-2, B-cell lymphoma-extra large (Bcl-xl), the p53 upregulated modulator of apoptosis (PUMA), cytochrome c, cleaved caspase 9, cleaved caspase 3, phospho-(p-)PI3K (Tyr458), PI3K, p-p38 (Thr180/Tyr182), p38, p-Akt (Ser473), Akt, p-mTOR (Ser2448), mTOR, beclin 1, microtubule-associated protein 1A/1B-light chain 3 (LC3-I), and LC3-II were all purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

    Techniques: Concentration Assay, Expressing

    Pro-inflammatory cytokines stimulate the AMPK-ULK-1 axis while inhibiting mTORC1 in β-cells a–d Prevalence of apoptosis was evaluated by HO-PI staining in INS-1E cells a, c or primary rat islets b, d treated or not (ctrl) for 16 h with IL-1β + IFNγ (cyt), alone or in combination with chloroquine (CQ; 10 µM), Bafilomycin A1 (Baf; 100 nM), 3-Methyladenine (3-MA, 5mM), rapamycin (rap; 100 nM), or torin1 (1–100 nM, as indicated). Data are mean ± SEM of 4–6 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: Dysfunctional autophagy following exposure to pro-inflammatory cytokines contributes to pancreatic β-cell apoptosis

    doi: 10.1038/s41419-017-0121-5

    Figure Lengend Snippet: Pro-inflammatory cytokines stimulate the AMPK-ULK-1 axis while inhibiting mTORC1 in β-cells a–d Prevalence of apoptosis was evaluated by HO-PI staining in INS-1E cells a, c or primary rat islets b, d treated or not (ctrl) for 16 h with IL-1β + IFNγ (cyt), alone or in combination with chloroquine (CQ; 10 µM), Bafilomycin A1 (Baf; 100 nM), 3-Methyladenine (3-MA, 5mM), rapamycin (rap; 100 nM), or torin1 (1–100 nM, as indicated). Data are mean ± SEM of 4–6 independent experiments. * P

    Article Snippet: The following chemicals were dissolved in DMSO and used as indicated: A23187 (Sigma; C7522; 1 μM), Tg (Tocris Bioscience; 1138; 100 nM), tunicamycin (Sigma; T7765; 5 µg/ml), torin1 (Tocris Bioscience; 4247; 1–100 nM), Bafilomycin A1 (Sigma; B1793; 100 nM), rapamycin (Calbiochem | 553210—Merck Millipore; 100 nM).

    Techniques: Staining

    Influence of autophagy stimulator or inhibitors on aurantiamide acetate-induced cell death. U87 cells were incubated with 25 μM aurantiamide acetate (AA), 10 nM rapamycin (Rapa), 10 mM 3-methyladenine (3-MA), 40 nM Bafilomycin A1 (BafA1), 10 μM chloroquine (CQ), 25 μM AA plus 10 nM Rapa, 25 μM AA plus 10 mM 3-MA, 25 μM AA plus 40 nM BafA1, or 25 μM AA plus 10 μM CQ. Cells without drug treatment were used as control (Ctrl). (A) Representative images were presented; scale bar, 50 μm. (B) The number of processes per cell was quantified from six micrographs. At least 30 cells per micrograph were analysed. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Aurantiamide acetate suppresses the growth of malignant gliomas in vitro and in vivo by inhibiting autophagic flux

    doi: 10.1111/jcmm.12498

    Figure Lengend Snippet: Influence of autophagy stimulator or inhibitors on aurantiamide acetate-induced cell death. U87 cells were incubated with 25 μM aurantiamide acetate (AA), 10 nM rapamycin (Rapa), 10 mM 3-methyladenine (3-MA), 40 nM Bafilomycin A1 (BafA1), 10 μM chloroquine (CQ), 25 μM AA plus 10 nM Rapa, 25 μM AA plus 10 mM 3-MA, 25 μM AA plus 40 nM BafA1, or 25 μM AA plus 10 μM CQ. Cells without drug treatment were used as control (Ctrl). (A) Representative images were presented; scale bar, 50 μm. (B) The number of processes per cell was quantified from six micrographs. At least 30 cells per micrograph were analysed. * P

    Article Snippet: 3-methyladenine (M9281) and chloroquine (CQ; C6628) were bought from Sigma-Aldrich (Shanghai) Trading Co., Ltd, Shanghai, China.

    Techniques: Incubation

    MAGI1 Knockdown functions through Wnt/β-catenin and PTEN/AKT signaling pathway in LN229 (PTEN wild-type) and glioma stem cells (GSCs). ( A ) The protein expression levels of β-catenin, cyclin D1, PTEN and p-AKT were measured by Western blotting in LN229 and GSCs. ( B ) The ratios of β-catenin, cyclin D1, PTEN and p-AKT were determined by giving a mean net density in LN229 and GSCs. Data were presented as the means ± standard errors. ** p

    Journal: OncoTargets and therapy

    Article Title: Silencing Of MAGI1 Promotes The Proliferation And Inhibits Apoptosis Of Glioma Cells Via The Wnt/β-Catenin And PTEN/AKT Signaling Pathways

    doi: 10.2147/OTT.S215400

    Figure Lengend Snippet: MAGI1 Knockdown functions through Wnt/β-catenin and PTEN/AKT signaling pathway in LN229 (PTEN wild-type) and glioma stem cells (GSCs). ( A ) The protein expression levels of β-catenin, cyclin D1, PTEN and p-AKT were measured by Western blotting in LN229 and GSCs. ( B ) The ratios of β-catenin, cyclin D1, PTEN and p-AKT were determined by giving a mean net density in LN229 and GSCs. Data were presented as the means ± standard errors. ** p

    Article Snippet: Immunoblotting was performed according to the standard protocol and the proteins were transferred to polyvinylidene membranes (Millipore, Billerica, MA, USA), blocked with TBST with 5% skimmed milk for 2 h at room temperature, and then incubated at 4°C overnight with either anti-MAGI1, anti-E-cadherin, anti-N-cadherin, anti-Bax, anti-Bcl2, anti-caspase3 (all 1:1,000, Abcam, Cambridge, MA, UK), anti-vimentin, anti-PTEN, anti phospho-Akt, anti β-catenin, anti-cyclin D1 (all 1:1,000, Cell Signaling Technology, Beverly MA, USA), or anti-GAPDH antibodies (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot

    The diagram of the cell signaling pathway which was involved in NVU protection of XXMD against cerebral injury following focal cerebral ischemia and reperfusion. Reperfusion after stroke induced dysfunction of PI3K/Akt pathway and the PI3K/Akt inhibition led to dephosphorylation of PDK1, Akt, and GSK3 β degradation, resulting in apoptosis. However, XXMD treatment inhibited apoptosis of different cells in NVU and increased expression levels of p-PTEN, p-PDK1, p-Akt, and p-GSK3 β . Furthermore, PI3K/Akt pathway is associated with Ras/MAPK pathway. In the present study, we also found that XXMD upregulated the level of p-c-raf. Above all, all the observations in the study indicated that XXMD may exert its NVU protection partly through the activation of PI3K/Akt signaling pathway.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: PI3K/Akt Pathway Contributes to Neurovascular Unit Protection of Xiao-Xu-Ming Decoction against Focal Cerebral Ischemia and Reperfusion Injury in Rats

    doi: 10.1155/2013/459467

    Figure Lengend Snippet: The diagram of the cell signaling pathway which was involved in NVU protection of XXMD against cerebral injury following focal cerebral ischemia and reperfusion. Reperfusion after stroke induced dysfunction of PI3K/Akt pathway and the PI3K/Akt inhibition led to dephosphorylation of PDK1, Akt, and GSK3 β degradation, resulting in apoptosis. However, XXMD treatment inhibited apoptosis of different cells in NVU and increased expression levels of p-PTEN, p-PDK1, p-Akt, and p-GSK3 β . Furthermore, PI3K/Akt pathway is associated with Ras/MAPK pathway. In the present study, we also found that XXMD upregulated the level of p-c-raf. Above all, all the observations in the study indicated that XXMD may exert its NVU protection partly through the activation of PI3K/Akt signaling pathway.

    Article Snippet: Reagents LY294002 (PI3K inhibitor), the primary antibodies for phospho-PDK1 (p-PDK1, Ser241), total PDK1, phospho-PTEN (p-PTEN, Ser380), total PTEN, phospho-Akt (p-Akt, Ser473), total Akt, phospho-c-Raf (p-c-Raf, Ser259), total c-Raf, phospho-GSK3β (p-GSK3β , Ser9), total GSK3β , GAPDH, and horseradish-peroxidase- (HRP-) linked anti-rabbit antibody purchased from Cell Signaling Technology (Beverly, MA, USA) were used for Western blot analysis.

    Techniques: Inhibition, De-Phosphorylation Assay, Expressing, Activation Assay

    Western blot analysis of phosphorylation levels of Akt, GSK3 β , and c-Raf at 24 h after reperfusion. (a) Representative protein bands for p-Akt (Ser473), total Akt. p-Akt (Ser473) expression levels significantly were decreased at 24 h after reperfusion, whereas XXMD treatment enhanced p-Akt (Ser473) levels and the effects could be partly reversed by PI3K inhibitor. No changes in Akt were detected in rats of different groups. GAPDH was used to show equal protein loading of each lane. (b) Representative protein bands for p-GSK3 β (Ser9), total GSK3 β . A decrease in p-GSK3 β (Ser9) level was observed in the peripheral area of ischemia after reperfusion and the levels of p-GSK3 β (Ser9) in the XXMD60 group were higher than those in the I/R group. However, inhibition of PI3K using LY294002 abolished the increase. No changes in GSK3 β were observed in rats of different groups. (c) Representative protein bands for p-c-Raf, total c-Raf. Although p-c-Raf expression levels significantly were decreased at 24 h after reperfusion, XXMD preserved the levels of p-c-Raf. Notably, LY294002 did not significantly block the effect of XXMD on p-c-Raf. No changes in c-Raf were detected in rats of different groups. Data are reported as the means ± SEM. n = 5; * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: PI3K/Akt Pathway Contributes to Neurovascular Unit Protection of Xiao-Xu-Ming Decoction against Focal Cerebral Ischemia and Reperfusion Injury in Rats

    doi: 10.1155/2013/459467

    Figure Lengend Snippet: Western blot analysis of phosphorylation levels of Akt, GSK3 β , and c-Raf at 24 h after reperfusion. (a) Representative protein bands for p-Akt (Ser473), total Akt. p-Akt (Ser473) expression levels significantly were decreased at 24 h after reperfusion, whereas XXMD treatment enhanced p-Akt (Ser473) levels and the effects could be partly reversed by PI3K inhibitor. No changes in Akt were detected in rats of different groups. GAPDH was used to show equal protein loading of each lane. (b) Representative protein bands for p-GSK3 β (Ser9), total GSK3 β . A decrease in p-GSK3 β (Ser9) level was observed in the peripheral area of ischemia after reperfusion and the levels of p-GSK3 β (Ser9) in the XXMD60 group were higher than those in the I/R group. However, inhibition of PI3K using LY294002 abolished the increase. No changes in GSK3 β were observed in rats of different groups. (c) Representative protein bands for p-c-Raf, total c-Raf. Although p-c-Raf expression levels significantly were decreased at 24 h after reperfusion, XXMD preserved the levels of p-c-Raf. Notably, LY294002 did not significantly block the effect of XXMD on p-c-Raf. No changes in c-Raf were detected in rats of different groups. Data are reported as the means ± SEM. n = 5; * P

    Article Snippet: Reagents LY294002 (PI3K inhibitor), the primary antibodies for phospho-PDK1 (p-PDK1, Ser241), total PDK1, phospho-PTEN (p-PTEN, Ser380), total PTEN, phospho-Akt (p-Akt, Ser473), total Akt, phospho-c-Raf (p-c-Raf, Ser259), total c-Raf, phospho-GSK3β (p-GSK3β , Ser9), total GSK3β , GAPDH, and horseradish-peroxidase- (HRP-) linked anti-rabbit antibody purchased from Cell Signaling Technology (Beverly, MA, USA) were used for Western blot analysis.

    Techniques: Western Blot, Expressing, Inhibition, Blocking Assay

    FPL64176 upregulates PKCα/βII, ERK1/2 and Akt phosphorylation in brainstem in a LTCC antagonist nifedipine-sensitive manner A–B) Time-courses of FPL64176-induced PKCα/βII, ERK1/2 and Akt phosphorylation the least shrew brainstem. Shrews (n =3 per group) were injected with either vehicle (0 min) or FPL 64176 (10 mg/kg., i.p.). Brainstems were collected at 0, 5, 10, 15, 20, 25, 30 and 60 min. Phospho-PKCα/βII at Thr638/641 (pPKCα/βII), GAPDH, phospho-ERK1/2 at Thr202/204 (pERK1/2), ERK1/2, phospho-Akt at Ser473 (pAkt) and Akt of protein samples extracted from individual brainstems were determined by Western blots. Panel A shows representative blots. Panel B shows summarized data. Bands were quantified using ImageJ software. Ratios of pPKCα/βII (~ 80 kD) to GAPDH (~ 37 kD), pERK1/2 (42/44 kD) to ERK1/2 and pAkt (~ 60 kD) to Akt were calculated and expressed as fold change (mean ± S.E.M.) of control (0 min). * P

    Journal: European journal of pharmacology

    Article Title: Intracellular emetic signaling evoked by the L-type Ca2+ channel agonist FPL64176 in the least shrew (Cryptotis parva)

    doi: 10.1016/j.ejphar.2018.06.035

    Figure Lengend Snippet: FPL64176 upregulates PKCα/βII, ERK1/2 and Akt phosphorylation in brainstem in a LTCC antagonist nifedipine-sensitive manner A–B) Time-courses of FPL64176-induced PKCα/βII, ERK1/2 and Akt phosphorylation the least shrew brainstem. Shrews (n =3 per group) were injected with either vehicle (0 min) or FPL 64176 (10 mg/kg., i.p.). Brainstems were collected at 0, 5, 10, 15, 20, 25, 30 and 60 min. Phospho-PKCα/βII at Thr638/641 (pPKCα/βII), GAPDH, phospho-ERK1/2 at Thr202/204 (pERK1/2), ERK1/2, phospho-Akt at Ser473 (pAkt) and Akt of protein samples extracted from individual brainstems were determined by Western blots. Panel A shows representative blots. Panel B shows summarized data. Bands were quantified using ImageJ software. Ratios of pPKCα/βII (~ 80 kD) to GAPDH (~ 37 kD), pERK1/2 (42/44 kD) to ERK1/2 and pAkt (~ 60 kD) to Akt were calculated and expressed as fold change (mean ± S.E.M.) of control (0 min). * P

    Article Snippet: The following primary antibodies were used for Western blot: phospho-ERK1/2 (1:1000, #9101, Cell Signaling, Danvers, MA), ERK1/2 (1:3000, #9107, Cell Signaling), phospho-Akt (Ser473) (1:2000, #4060, Cell Signaling), Akt (1:2000, #2920, Cell Signaling), phospho-PKCα/βII (Thr638/641) (1:1000, #9375, Cell Signaling), and GAPDH (1:10000, MAB374, EMD Millipore, Temecula, CA).

    Techniques: Injection, Western Blot, Software

    In vitro  release of plumbagin from oleic acid-based nanoemulsion (polysorbate 80 (3.5%) as a function of time. SGF, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) at 37°C was used as release media.

    Journal: BioMed Research International

    Article Title: Plumbagin-Loaded Nanoemulsion Drug Delivery Formulation and Evaluation of Antiproliferative Effect on Prostate Cancer Cells

    doi: 10.1155/2018/9035452

    Figure Lengend Snippet: In vitro release of plumbagin from oleic acid-based nanoemulsion (polysorbate 80 (3.5%) as a function of time. SGF, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) at 37°C was used as release media.

    Article Snippet: Plumbagin, polysorbate 80, oleic acid and all other chemicals were supplied by Sigma (St. Louis, MO, USA) unless otherwise stated.

    Techniques: In Vitro

    Changes in expression of antioxidant enzyme, oxidative stress marker, mitochondrial activity, and proteins involved in mitochondrial function after exposure to ozone in IL-1α-pretreated conjunctival epithelial cells. (A) Antioxidant enzyme and oxidative stress marker. (B) Mitochondrial activity and proteins involved in mitochondrial function. IL, interleukin; Mn-SOD, manganese superoxide dismutase; TRXr-1, thioredoxin reductase-1; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; mTOR, mammalian target of rapamycin; p-mTOR, phospho-mammalian target of rapamycin. Error bars represent the standard error of the mean (* p

    Journal: PLoS ONE

    Article Title: Effects of Exposure to Ozone on the Ocular Surface in an Experimental Model of Allergic Conjunctivitis

    doi: 10.1371/journal.pone.0169209

    Figure Lengend Snippet: Changes in expression of antioxidant enzyme, oxidative stress marker, mitochondrial activity, and proteins involved in mitochondrial function after exposure to ozone in IL-1α-pretreated conjunctival epithelial cells. (A) Antioxidant enzyme and oxidative stress marker. (B) Mitochondrial activity and proteins involved in mitochondrial function. IL, interleukin; Mn-SOD, manganese superoxide dismutase; TRXr-1, thioredoxin reductase-1; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; mTOR, mammalian target of rapamycin; p-mTOR, phospho-mammalian target of rapamycin. Error bars represent the standard error of the mean (* p

    Article Snippet: The membranes were blocked overnight at 4°C in 5% bovine serum albumin or 5% nonfat dry milk in a buffer containing 10 mM Tris-HCl (pH 8.0) (Sigma-Aldrich), 150 mM NaCl, and 0.05% Tween-20 (Sigma-Aldrich), and then incubated overnight with the primary antibodies for manganese superoxide dismutase (Mn-SOD; Stressgen, Victoria, BC, Canada), catalase (Abcam, Cambridge, UK), thioredoxin reductase-1 (TRXr-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA), heme-oxygenase-1 (HO-1; Santa Cruz Biotechnology), total OXPHOS Complexes Detection Kit (Mitosciences Inc., Eugene, OR, USA), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α; Novus Biologicals, LLC, Littleton, CO, USA), voltage-dependent anion channel (VDAC; Cell Signaling Technology, Beverly, MA, USA), mammalian target of rapamycin (mTOR), phospho-mTOR (p-mTOR, Cell Signaling Technology), Akt, p-Akt (Cell Signaling Technology), Lamin A/C (Cell Signaling Technology), or β-actin (Santa Cruz Biotechnology), followed by incubation with peroxidase-labeled anti-mouse IgG secondary antibody (KPL Laboratories, Gaithersburg, MD, USA) or peroxidase-labeled anti-rabbit IgG secondary antibody (KPL Laboratories). β-actin served as a loading control.

    Techniques: Expressing, Marker, Activity Assay, Pyrolysis Gas Chromatography

    The mTOR/S6K1 signaling pathway is not activated in response to the oxidative stress produced by H 2 O 2 . MCF7 cells were transfected with the indicated siRNAs 72 h before treatment with 0.4 mM H 2 O 2 for 30 min. Cell lysates were analyzed by Western blot with the indicated antibodies. Molecular weight markers are indicated on the left. NT means non-targeting control.

    Journal: PLoS ONE

    Article Title: Contribution of S6K1/MAPK Signaling Pathways in the Response to Oxidative Stress: Activation of RSK and MSK by Hydrogen Peroxide

    doi: 10.1371/journal.pone.0075523

    Figure Lengend Snippet: The mTOR/S6K1 signaling pathway is not activated in response to the oxidative stress produced by H 2 O 2 . MCF7 cells were transfected with the indicated siRNAs 72 h before treatment with 0.4 mM H 2 O 2 for 30 min. Cell lysates were analyzed by Western blot with the indicated antibodies. Molecular weight markers are indicated on the left. NT means non-targeting control.

    Article Snippet: Reagents Insulin, wortmannin, rapamycin and anti-P-ERK1/2 antibody (Sigma-Aldrich); hydrogen peroxide solution (H2 O2 ) (Panreac); U0126 and SB203580 (Calbiochem); anti-mTOR, anti-P-T389-S6K1 (1A5), anti-P-S380-RSK, anti-P-S376-MSK, anti-P-S235/236-S6, anti-S6 (54D2) and anti-PT180/Y182-p38 antibodies (Cell Signaling Technology); anti-MSK, anti-S6K1 (C-18) and anti-RSK1 (C-21) antibodies (Santa Cruz Biotechnology, Inc.); Alexa Fluor 488, Alexa Fluor 546, TO-PRO3 (Molecular Probes); anti-P- H2AX antibody, Immobilon-P PVDF transfer membrane (Millipore Corporation); siRNA used: mTOR (CCCUGCCUUUGUCAUGCCUdTdT), S6K1 (GGGGGCUAUGGAAAGGUUUdTdT), RSK1 ( GCUAUACCGUCGUGA -GAUCdTdT), RSK2 (GGAGGAGAUUAACCCACAAdTdT), MSK1 ( GGAACUGG -AGCUUAUGGAAdTdT), MSK2 (UUGCACAUGAUCUCGGCCGdTdT) and non-targeting control (UAGCGACUAAACACAUCAAdTdT).

    Techniques: Produced, Transfection, Western Blot, Molecular Weight

    Phosphorylation of p85, RSK and MSK proteins was sensitive to inhibitors of the MAPK signaling pathways. (A) MCF7 cells were treated with 0.4 mM H 2 O 2 for 30 min. Where indicated, cells were pre-incubated with 5 µM SB203580 (S), 5 µM U0126 (U), 100 nM wortmannin (W) or 20 nM rapamycin (R) for 60 min before treatment with H 2 O 2 . Cell lysates were analyzed by Western blot with the indicated antibodies. NSB means non-specific band recognized by the antibody. Molecular weight markers are indicated on the left. (B) Histograms represent the phosphorylation ratio of the indicated proteins. All bands were standardized with respect to mTOR levels. Values are the means ± SEM of the percentage of respective control for at least three independent experiments. Asterisks indicate values that are significantly different (*, p

    Journal: PLoS ONE

    Article Title: Contribution of S6K1/MAPK Signaling Pathways in the Response to Oxidative Stress: Activation of RSK and MSK by Hydrogen Peroxide

    doi: 10.1371/journal.pone.0075523

    Figure Lengend Snippet: Phosphorylation of p85, RSK and MSK proteins was sensitive to inhibitors of the MAPK signaling pathways. (A) MCF7 cells were treated with 0.4 mM H 2 O 2 for 30 min. Where indicated, cells were pre-incubated with 5 µM SB203580 (S), 5 µM U0126 (U), 100 nM wortmannin (W) or 20 nM rapamycin (R) for 60 min before treatment with H 2 O 2 . Cell lysates were analyzed by Western blot with the indicated antibodies. NSB means non-specific band recognized by the antibody. Molecular weight markers are indicated on the left. (B) Histograms represent the phosphorylation ratio of the indicated proteins. All bands were standardized with respect to mTOR levels. Values are the means ± SEM of the percentage of respective control for at least three independent experiments. Asterisks indicate values that are significantly different (*, p

    Article Snippet: Reagents Insulin, wortmannin, rapamycin and anti-P-ERK1/2 antibody (Sigma-Aldrich); hydrogen peroxide solution (H2 O2 ) (Panreac); U0126 and SB203580 (Calbiochem); anti-mTOR, anti-P-T389-S6K1 (1A5), anti-P-S380-RSK, anti-P-S376-MSK, anti-P-S235/236-S6, anti-S6 (54D2) and anti-PT180/Y182-p38 antibodies (Cell Signaling Technology); anti-MSK, anti-S6K1 (C-18) and anti-RSK1 (C-21) antibodies (Santa Cruz Biotechnology, Inc.); Alexa Fluor 488, Alexa Fluor 546, TO-PRO3 (Molecular Probes); anti-P- H2AX antibody, Immobilon-P PVDF transfer membrane (Millipore Corporation); siRNA used: mTOR (CCCUGCCUUUGUCAUGCCUdTdT), S6K1 (GGGGGCUAUGGAAAGGUUUdTdT), RSK1 ( GCUAUACCGUCGUGA -GAUCdTdT), RSK2 (GGAGGAGAUUAACCCACAAdTdT), MSK1 ( GGAACUGG -AGCUUAUGGAAdTdT), MSK2 (UUGCACAUGAUCUCGGCCGdTdT) and non-targeting control (UAGCGACUAAACACAUCAAdTdT).

    Techniques: Incubation, Western Blot, Molecular Weight

    Bortezomib (BTZ) induces the phosphorylation of ERK, reduces the level of cathepsin B (CTSB), and suppresses the catalytic process in autophagic lysosomes. ( a ) TOV112D cells were treated with 0.05 μ M BTZ for 24 h. CTSB protein and mRNA levels were measured by immunoblotting and real-time quantitative PCR, respectively. Results shown are mean±standard error for three independent experiments. ( b ) The effects of the overexpression of CTSB for 72 h on the TOV112D cells treated with 0.05 μ M BTZ for 24 h were analyzed by measuring the p62 protein level and ( c ) using the MTT assays. Forced expression of C9-CTSB was detected by anti-C9 antibody with western blot analysis. Results shown are the mean±standard error for three independent experiments. ( d ) The effects induced by the treatment with 0.05 μ M BTZ and 30 μ M PD98059 for 24 h were analyzed in TOV112D cells using immunoblotting for the levels of ERK phosphorylation, CTSB, and p62. ( e ) The effect of forced expression of a constitutively active p-ERK (Y204D) for 72 h in TOV112D cells were analyzed by measuring the level of CTSB. Results shown are mean ± standard error for three independent experiments. ( f ) TOV112D cells were transfected with a C9-CTSB expression vector and treated with 0.05 μ M BTZ. After lysosomal labeling with 300 nM Lysotracker (red) for 16 h, the colocalization of CTSB and functional lysosomes was shown by yellow merged signals of anti-C9 (green) into Lysotracker (red). Scale bar represents 30 μ m

    Journal: Cell Death & Disease

    Article Title: Bortezomib enhances cancer cell death by blocking the autophagic flux through stimulating ERK phosphorylation

    doi: 10.1038/cddis.2014.468

    Figure Lengend Snippet: Bortezomib (BTZ) induces the phosphorylation of ERK, reduces the level of cathepsin B (CTSB), and suppresses the catalytic process in autophagic lysosomes. ( a ) TOV112D cells were treated with 0.05 μ M BTZ for 24 h. CTSB protein and mRNA levels were measured by immunoblotting and real-time quantitative PCR, respectively. Results shown are mean±standard error for three independent experiments. ( b ) The effects of the overexpression of CTSB for 72 h on the TOV112D cells treated with 0.05 μ M BTZ for 24 h were analyzed by measuring the p62 protein level and ( c ) using the MTT assays. Forced expression of C9-CTSB was detected by anti-C9 antibody with western blot analysis. Results shown are the mean±standard error for three independent experiments. ( d ) The effects induced by the treatment with 0.05 μ M BTZ and 30 μ M PD98059 for 24 h were analyzed in TOV112D cells using immunoblotting for the levels of ERK phosphorylation, CTSB, and p62. ( e ) The effect of forced expression of a constitutively active p-ERK (Y204D) for 72 h in TOV112D cells were analyzed by measuring the level of CTSB. Results shown are mean ± standard error for three independent experiments. ( f ) TOV112D cells were transfected with a C9-CTSB expression vector and treated with 0.05 μ M BTZ. After lysosomal labeling with 300 nM Lysotracker (red) for 16 h, the colocalization of CTSB and functional lysosomes was shown by yellow merged signals of anti-C9 (green) into Lysotracker (red). Scale bar represents 30 μ m

    Article Snippet: Rapamycin (Sigma, R8781-200UL) was dissolved in DMSO at 10 mM, and PD98059 (Sigma, P215-1MG) was dissolved in DMSO at 100 mM.

    Techniques: Real-time Polymerase Chain Reaction, Over Expression, MTT Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Labeling, Functional Assay

    The involvement of AMPK in insulin-induced vasodilation in the presence of PVAT through the secretion of adiponectin. A : The AMPK inhibitor Compound C (Cmpd C) inhibits the insulin-induced vasodilation nonsignificantly in the presence of C57BL/6 PVAT (black square, C57BL/6 resistance artery [RA], with C57BL/6 PVAT [ n = 10]; black circle, C57BL/6 RA in the presence of C57BL/6 PVAT and compound C [ n = 7]). B : PVAT from C57BL/6 mice induces significant threonine 172 phosphorylation of AMPK, as does PVAT from db / db mice. Threonine 172 phosphorylation of AMPK was, however, not significantly different between the two types of PVAT. * P

    Journal: Diabetes

    Article Title: Perivascular Adipose Tissue Control of Insulin-Induced Vasoreactivity in Muscle Is Impaired in db/db Mice

    doi: 10.2337/db11-1603

    Figure Lengend Snippet: The involvement of AMPK in insulin-induced vasodilation in the presence of PVAT through the secretion of adiponectin. A : The AMPK inhibitor Compound C (Cmpd C) inhibits the insulin-induced vasodilation nonsignificantly in the presence of C57BL/6 PVAT (black square, C57BL/6 resistance artery [RA], with C57BL/6 PVAT [ n = 10]; black circle, C57BL/6 RA in the presence of C57BL/6 PVAT and compound C [ n = 7]). B : PVAT from C57BL/6 mice induces significant threonine 172 phosphorylation of AMPK, as does PVAT from db / db mice. Threonine 172 phosphorylation of AMPK was, however, not significantly different between the two types of PVAT. * P

    Article Snippet: As a model to inhibit downstream adiponectin signaling, the effects of the AMPK inhibitor Compound C (1 μmol/L; product number 171260; Calbiochem) on insulin-induced vasoreactivity were examined in C57BL/6 resistance arteries in the presence of C57BL/6 PVAT (n = 7).

    Techniques: Mouse Assay

    Top : Akt Ser 473 phosphorylation (pAkt Ser473 ) in epitrochlearis and soleus muscles with 0, 1.2, or 30 nM insulin. Middle : pAkt1 Ser473 in epitrochlearis and soleus muscles with 0, 1.2, or 30 nM insulin. Muscle lysate was immunoprecipitated with Akt1 antibody

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats

    doi: 10.1152/ajpendo.00659.2010

    Figure Lengend Snippet: Top : Akt Ser 473 phosphorylation (pAkt Ser473 ) in epitrochlearis and soleus muscles with 0, 1.2, or 30 nM insulin. Middle : pAkt1 Ser473 in epitrochlearis and soleus muscles with 0, 1.2, or 30 nM insulin. Muscle lysate was immunoprecipitated with Akt1 antibody

    Article Snippet: Anti-Appl1 (catalog no. ab59592) was obtained from Abcam (Cambridge, MA); anti-phospho-AS160 Ser588 (pAS160Ser588 ; catalog no. 3028P2) from B-Bridge International (Mountain View, CA); anti-PP2A (catalog no. 610555) from BD Biosciences (San Jose, CA); anti-Akt (catalog no. 9272), anti-5′-AMP-activated protein kinase subunit-α (AMPKα; catalog no. 2532), anti-PKCζ (catalog no. 9372), anti-phospho-Akt (Ser/Thr) substrate (PAS; catalog no. 9611), anti-phospho-Akt Thr308 (pAktThr308 ; catalog no. 9275), anti-phospho-Akt Ser473 (pAktSer473 ; catalog no. 9272), anti-phospho-AMPKα Thr172 (pAMPKThr172 ; catalog no. 2531), anti-GLUT4 (catalog no. 2299), anti-Grb2 (catalog no. 3972), and anti-rabbit IgG-horseradish peroxidase conjugate (catalog no. 7074) from Cell Signaling Technology (Danvers, MA); anti-HSP90 (catalog no. ADI-SPA-846) from Enzo Life Sciences (Plymouth Meeting, PA); anti-phospho-IR Tyr1162/1163 (pIRTyr1162/1163 ; catalog no. 44-504G) and anti-IR (catalog no. AHR0271) from Invitrogen (Camarillo, CA); anti-phospho-AS160 Thr642 (pAS160Thr642 ; catalog no. 07-802), anti-AS160 (catalog no. 07-741), anti-mouse IgG-horseradish peroxidase conjugate (catalog no. 12-349), and anti-sheep IgG horseradish peroxidase conjugate (catalog no. 12-342) from Millipore (Billerica, MA); anti-Akt2 (catalog no. AF23151) from R & D Biosystems (Minneapolis, MN); and anti-Akt1 (catalog no. sc-7126), anti-aPKCζ/λ (catalog no. sc-216), and anti-mouse IgG-horseradish peroxidase conjugate (catalog no. sc-2060) from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Immunoprecipitation

    Regulation of TcADKn nucleolar localization. A) Fluorescence microscopy of T. cruzi epimastigotes in exponential growth phase, treated with Control (control), actinomycin D 10 µg.mL −1 for 4 h, (ActD), puromycin 200 µg.mL −1 for 4 h (Puro), cycloheximide 50 µg.mL −1 for 4 h (CHC), rapamycin 100 µM 12 h (Rapa), hydrogen peroxide 200 µM for 1 h (H 2 O 2 ) and phleomycin 100 µg.mL −1 1 hour (Phleo). Drugs were provided by Sigma. B) For rapamycin treatment, fluorescence was quantified for 40 treated and untreated parasites. In each parasite the fluorescence from the green channel (GFP) was quantified in an area selected according to blue signal (DAPI) fluorescence (nucleus) using the RGB plugin in ImageJ ( http://rsb.info.nih.gov/ij ). Cytoplasmic fluorescence was quantified in the same way selecting the brightest perinuclear areas in the green channel (GFP). Selection criterion was the same for all transfected parasites. Bars represent mean ± S.D. Statistically significant difference was calculated by t-student test (p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform

    doi: 10.1371/journal.pntd.0002044

    Figure Lengend Snippet: Regulation of TcADKn nucleolar localization. A) Fluorescence microscopy of T. cruzi epimastigotes in exponential growth phase, treated with Control (control), actinomycin D 10 µg.mL −1 for 4 h, (ActD), puromycin 200 µg.mL −1 for 4 h (Puro), cycloheximide 50 µg.mL −1 for 4 h (CHC), rapamycin 100 µM 12 h (Rapa), hydrogen peroxide 200 µM for 1 h (H 2 O 2 ) and phleomycin 100 µg.mL −1 1 hour (Phleo). Drugs were provided by Sigma. B) For rapamycin treatment, fluorescence was quantified for 40 treated and untreated parasites. In each parasite the fluorescence from the green channel (GFP) was quantified in an area selected according to blue signal (DAPI) fluorescence (nucleus) using the RGB plugin in ImageJ ( http://rsb.info.nih.gov/ij ). Cytoplasmic fluorescence was quantified in the same way selecting the brightest perinuclear areas in the green channel (GFP). Selection criterion was the same for all transfected parasites. Bars represent mean ± S.D. Statistically significant difference was calculated by t-student test (p

    Article Snippet: Drug and differential media treatments Exponentially growing T. cruzi epimastigotes were treated with different drugs: actinomycin D (Sigma) 10 µg.mL−1 for 4 h, cicloheximide (Sigma) 50 µg.mL−1 for 4 h, puromycin 200 µg.mL−1 4 h, starvation in PBS 24 h, leptomycin B (Sigma) 0.1 µg.mL−1 for 5 h, rapamycin (Sigma) 100 µM for 6–8 h, phleomycin 150 µg.mL−1 for 4 h, hydrogen peroxide 200 µM for 1 h. After leptomycin and rapamycin treatment fluorescence was quantified for forty treated and untreated parasites.

    Techniques: Fluorescence, Microscopy, Selection, Transfection

    Regulation of eEF2K in cells expressing BOP1Δ. ( A ) T-REx cells were treated for 15 h with 1 µg/ml doxycycline and then for 1 h with 20 µg of MG132. Cell lysates were subjected to western blot. ( B ) T-REx cells were treated for 15 h with 1 µg/ml doxycycline and treated with PP242 (1 µM, 15 h). Total RNA was extracted and subjected to RT-qPCR analysis for eEF2K and β-actin mRNAs. The significance was determined by t -test. ( C ) T-REx cells expressing BOP1Δ were cultured in complete medium with/without doxycycline. After 33 h, in some cases, cells were treated with 1 µM PP242 or 100 nM rapamycin for 15 h. Cells were lysed and 20 µg of lysate proteins were used for western blot analysis. ( D ) T-REx cells inducibly expressing BOP1Δ were cultured in complete medium with/without doxycycline. After 36 h, in some cases, cells were treated overnight with 10 µM PF-4708671 or 100 nM rapamycin. Cells were lysed and 20 µg of protein were used for western blot analysis or to measure the activity of eEF2K using eEF2 and [γ- 32 P]ATP as substrates.

    Journal: Nucleic Acids Research

    Article Title: Impairing the production of ribosomal RNA activates mammalian target of rapamycin complex 1 signalling and downstream translation factors

    doi: 10.1093/nar/gku130

    Figure Lengend Snippet: Regulation of eEF2K in cells expressing BOP1Δ. ( A ) T-REx cells were treated for 15 h with 1 µg/ml doxycycline and then for 1 h with 20 µg of MG132. Cell lysates were subjected to western blot. ( B ) T-REx cells were treated for 15 h with 1 µg/ml doxycycline and treated with PP242 (1 µM, 15 h). Total RNA was extracted and subjected to RT-qPCR analysis for eEF2K and β-actin mRNAs. The significance was determined by t -test. ( C ) T-REx cells expressing BOP1Δ were cultured in complete medium with/without doxycycline. After 33 h, in some cases, cells were treated with 1 µM PP242 or 100 nM rapamycin for 15 h. Cells were lysed and 20 µg of lysate proteins were used for western blot analysis. ( D ) T-REx cells inducibly expressing BOP1Δ were cultured in complete medium with/without doxycycline. After 36 h, in some cases, cells were treated overnight with 10 µM PF-4708671 or 100 nM rapamycin. Cells were lysed and 20 µg of protein were used for western blot analysis or to measure the activity of eEF2K using eEF2 and [γ- 32 P]ATP as substrates.

    Article Snippet: Chemicals and antibodies Doxycycline was purchased from Fisher, MG132 from Calbiochem, rapamycin from Merck, PP242 from Sigma-Aldrich, AZD6244 from Selleck and PF4708671 from Tocris.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Cell Culture, Activity Assay

    MCF7 Snail-6SA has increased Erk phosphorylation and decreased sensitivity to higher doses of rapamycin A. Snail, E-cadherin and vimentin expression was assessed in stably transfected MCF7 snail wild-type (Sn-WT) and MCF7 snail mutant (Sn-2SA and Sn-6SA) cell lines by western blotting. B. MCF7 snail wild-type (Snail-WT) and mutant (Snail-6SA) cell lines were treated with DMSO 0.1% or rapamycin 100 nM daily for 3 days. EMT, MAPK and mTOR pathway markers were assessed by western blotting. C. Rapamycin sensitivity in MCF7 snail wild-type (Snail-WT) or mutant (Snail-6SA) cell lines were assessed by SRB assay following 96-hour treatment with increasing doses of rapamycin. ** p

    Journal: Oncotarget

    Article Title: Epithelial to mesenchymal transition is associated with rapamycin resistance

    doi:

    Figure Lengend Snippet: MCF7 Snail-6SA has increased Erk phosphorylation and decreased sensitivity to higher doses of rapamycin A. Snail, E-cadherin and vimentin expression was assessed in stably transfected MCF7 snail wild-type (Sn-WT) and MCF7 snail mutant (Sn-2SA and Sn-6SA) cell lines by western blotting. B. MCF7 snail wild-type (Snail-WT) and mutant (Snail-6SA) cell lines were treated with DMSO 0.1% or rapamycin 100 nM daily for 3 days. EMT, MAPK and mTOR pathway markers were assessed by western blotting. C. Rapamycin sensitivity in MCF7 snail wild-type (Snail-WT) or mutant (Snail-6SA) cell lines were assessed by SRB assay following 96-hour treatment with increasing doses of rapamycin. ** p

    Article Snippet: DMSO (vehicle for rapamycin for in vitro and in vivo experiments), polyethylene glycol (vehicle for vorinostat for in vivo experiments), and G418 were purchased from Sigma.

    Techniques: Expressing, Stable Transfection, Transfection, Mutagenesis, Western Blot, Sulforhodamine B Assay

    Trametinib modulated EMT in ACHN and increased sensitivity of ACHN to lower doses of rapamycin A. ACHN (left) and MDA-MB-231 (right) were treated daily with DMSO 0.1% (D), rapamycin 10 nM (R10), rapamycin 100 nM (R100), trametinib 10 μM (T), or in combination with each rapamycin dose (T+R10, T+R100) for 3 days and harvested for western blotting 24 hours later to assess EMT, MAPK and mTOR signaling. B. ACHN xenografts were treated with DMSO/Vehicle (Vehicle), rapamycin 1 mg/kg /vehicle (Rapamycin), DMSO/trametinib 0.3 mg/kg (Trametinib), rapamycin/trametinib (Rapa/Tram) at the same doses, or trametinib daily for three days then followed by rapamycin/trametinib (Del Rapa/Tram) at the same doses daily for a total of 14 days. * p

    Journal: Oncotarget

    Article Title: Epithelial to mesenchymal transition is associated with rapamycin resistance

    doi:

    Figure Lengend Snippet: Trametinib modulated EMT in ACHN and increased sensitivity of ACHN to lower doses of rapamycin A. ACHN (left) and MDA-MB-231 (right) were treated daily with DMSO 0.1% (D), rapamycin 10 nM (R10), rapamycin 100 nM (R100), trametinib 10 μM (T), or in combination with each rapamycin dose (T+R10, T+R100) for 3 days and harvested for western blotting 24 hours later to assess EMT, MAPK and mTOR signaling. B. ACHN xenografts were treated with DMSO/Vehicle (Vehicle), rapamycin 1 mg/kg /vehicle (Rapamycin), DMSO/trametinib 0.3 mg/kg (Trametinib), rapamycin/trametinib (Rapa/Tram) at the same doses, or trametinib daily for three days then followed by rapamycin/trametinib (Del Rapa/Tram) at the same doses daily for a total of 14 days. * p

    Article Snippet: DMSO (vehicle for rapamycin for in vitro and in vivo experiments), polyethylene glycol (vehicle for vorinostat for in vivo experiments), and G418 were purchased from Sigma.

    Techniques: Multiple Displacement Amplification, Western Blot

    Effect of rapamycin and STATTIC on cell proliferation and apoptosis in LCLs. ( a,b ) LCLs derived from both healthy donors ( a ) and SDS patients ( b ) were incubated in the presence or in the absence of 350 nM mTOR inhibitor rapamycin, or 20 μM STAT3 inhibitor STATTIC and stimulated with increasing doses (0.01–10 ng/ml) of IL-6 for 48 hours. Cell proliferation was measured by XTT Cell Proliferation Kit II. Data are mean ± SEM of five independent experiments performed in duplicate. Student’s t-test has been calculated. ( c,d ) LCLs derived from both healthy donors ( c ) and SDS patients ( d ) were incubated in the presence (black bars) or in the absence (white bars) of 350 nM mTOR inhibitor rapamycin for 24 hours. Apoptosis was analyzed using the Muse Annexin V Dead Cell Kit. Data are mean ± SEM of four independent experiments performed in duplicate. ( e,f ) LCLs derived from both healthy donors ( e ) and SDS patients ( f ) were incubated in the presence (black bars) or in the absence (white bars) of 20 μM STAT3 inhibitor STATTIC for 24 hours. Apoptosis was analyzed using the Muse Annexin V Dead Cell Kit. Data are mean ± SEM of four independent experiments performed in duplicate. Student’s t-test has been calculated.

    Journal: Scientific Reports

    Article Title: New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes

    doi: 10.1038/srep33165

    Figure Lengend Snippet: Effect of rapamycin and STATTIC on cell proliferation and apoptosis in LCLs. ( a,b ) LCLs derived from both healthy donors ( a ) and SDS patients ( b ) were incubated in the presence or in the absence of 350 nM mTOR inhibitor rapamycin, or 20 μM STAT3 inhibitor STATTIC and stimulated with increasing doses (0.01–10 ng/ml) of IL-6 for 48 hours. Cell proliferation was measured by XTT Cell Proliferation Kit II. Data are mean ± SEM of five independent experiments performed in duplicate. Student’s t-test has been calculated. ( c,d ) LCLs derived from both healthy donors ( c ) and SDS patients ( d ) were incubated in the presence (black bars) or in the absence (white bars) of 350 nM mTOR inhibitor rapamycin for 24 hours. Apoptosis was analyzed using the Muse Annexin V Dead Cell Kit. Data are mean ± SEM of four independent experiments performed in duplicate. ( e,f ) LCLs derived from both healthy donors ( e ) and SDS patients ( f ) were incubated in the presence (black bars) or in the absence (white bars) of 20 μM STAT3 inhibitor STATTIC for 24 hours. Apoptosis was analyzed using the Muse Annexin V Dead Cell Kit. Data are mean ± SEM of four independent experiments performed in duplicate. Student’s t-test has been calculated.

    Article Snippet: After this period, cells were seeded at a density of 5 × 104 cells/well in 96 multiwells plates in 100 μl/well RPMI-1640 medium containing 10% FBS in the presence or in the absence of 350 nM mTOR inhibitor Rapamycin (Sigma-Aldrich) or 10 μM STAT-3 inhibitor STATTIC (Sigma-Aldrich).

    Techniques: Derivative Assay, Incubation

    Model of dysregulated mTOR/STAT3 signal transduction pathways observed in leukocytes obtained from SDS patients. Normally, IL-6 trigger a JAK1/2 activation which in turn leads to STAT3 phosphorylation, mainly at Y705 residue, causing STAT3 dimerization and translocation into the nucleus, where STAT3 is able to regulate gene expression orchestrating several cellular processes like inflammation and cell proliferation. In SDS patients, leukocytes show ERK1/2, mTOR and STAT3 hyper-activation. ERK1/2 is known to promote mTOR phosphorylation in S2448 residue, which in turn leads to mTORC1 complex activation. mTORC1 is known to regulate different cell processes, including translation, autophagy, cell growth and ribosome biogenesis, which are impaired in SDS pathology. Notably, mTORC1 is also known to induce strong phosphorylation of STAT3 both in Y705 and S727 residues in different cellular models. Here we report that mTOR inhibitor rapamycin is able to reduce STAT3 hyper-activation observed in SDS patients, restoring phosphorylation level of both Y705 and S727. Moreover, in this issue we show how loss of SBDS protein can lead to mTOR S2448 hyper-activation in LCLs obtained from healthy donors. Finally, we show that pre-incubating ERK1/2 inhibitor U0126 in SDS EBV-transformed B cells we significantly reduce IL-6 induced mTOR S2448 phosphorylation.

    Journal: Scientific Reports

    Article Title: New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes

    doi: 10.1038/srep33165

    Figure Lengend Snippet: Model of dysregulated mTOR/STAT3 signal transduction pathways observed in leukocytes obtained from SDS patients. Normally, IL-6 trigger a JAK1/2 activation which in turn leads to STAT3 phosphorylation, mainly at Y705 residue, causing STAT3 dimerization and translocation into the nucleus, where STAT3 is able to regulate gene expression orchestrating several cellular processes like inflammation and cell proliferation. In SDS patients, leukocytes show ERK1/2, mTOR and STAT3 hyper-activation. ERK1/2 is known to promote mTOR phosphorylation in S2448 residue, which in turn leads to mTORC1 complex activation. mTORC1 is known to regulate different cell processes, including translation, autophagy, cell growth and ribosome biogenesis, which are impaired in SDS pathology. Notably, mTORC1 is also known to induce strong phosphorylation of STAT3 both in Y705 and S727 residues in different cellular models. Here we report that mTOR inhibitor rapamycin is able to reduce STAT3 hyper-activation observed in SDS patients, restoring phosphorylation level of both Y705 and S727. Moreover, in this issue we show how loss of SBDS protein can lead to mTOR S2448 hyper-activation in LCLs obtained from healthy donors. Finally, we show that pre-incubating ERK1/2 inhibitor U0126 in SDS EBV-transformed B cells we significantly reduce IL-6 induced mTOR S2448 phosphorylation.

    Article Snippet: After this period, cells were seeded at a density of 5 × 104 cells/well in 96 multiwells plates in 100 μl/well RPMI-1640 medium containing 10% FBS in the presence or in the absence of 350 nM mTOR inhibitor Rapamycin (Sigma-Aldrich) or 10 μM STAT-3 inhibitor STATTIC (Sigma-Aldrich).

    Techniques: Transduction, Activation Assay, Translocation Assay, Expressing, Transformation Assay

    Flow cytometric analysis of mTOR S2448 phosphorylation in LCLs. (a ) Representative experiment indicating mTOR S2448 phosphorylation level (green histogram) in healthy donor derived LCLs (Control) versus SDS LCLs (SDS). Red histogram indicates isotype control. Control LCLs and SDS LCLs were pre-incubated with 350 nM rapamycin (Rapa) for 1 hour before stimulation in the presence or in the absence (UT) of IL-6 (10 ng/ml) for further 15 min. ( b ) Median Fluorescence Intensity (MFI) and Percent of positive cells (c) derived from five independent experiments performed in LCLs derived from five different SDS patients. Data are mean ± SEM. Student’s t-test has been calculated.

    Journal: Scientific Reports

    Article Title: New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes

    doi: 10.1038/srep33165

    Figure Lengend Snippet: Flow cytometric analysis of mTOR S2448 phosphorylation in LCLs. (a ) Representative experiment indicating mTOR S2448 phosphorylation level (green histogram) in healthy donor derived LCLs (Control) versus SDS LCLs (SDS). Red histogram indicates isotype control. Control LCLs and SDS LCLs were pre-incubated with 350 nM rapamycin (Rapa) for 1 hour before stimulation in the presence or in the absence (UT) of IL-6 (10 ng/ml) for further 15 min. ( b ) Median Fluorescence Intensity (MFI) and Percent of positive cells (c) derived from five independent experiments performed in LCLs derived from five different SDS patients. Data are mean ± SEM. Student’s t-test has been calculated.

    Article Snippet: After this period, cells were seeded at a density of 5 × 104 cells/well in 96 multiwells plates in 100 μl/well RPMI-1640 medium containing 10% FBS in the presence or in the absence of 350 nM mTOR inhibitor Rapamycin (Sigma-Aldrich) or 10 μM STAT-3 inhibitor STATTIC (Sigma-Aldrich).

    Techniques: Flow Cytometry, Derivative Assay, Incubation, Fluorescence

    Autophagic Activity is Inhibited by Rhein through the MAPKs Signaling Pathways. ( A ) NRK-52E cells were treated with HBSS for 0, 0.5, 1, 2 and 6 hours and subjected to western blot analysis of the phosphorylation of p38 (p-p38), Erk (p-Erk) and JNK (p-JNK). ( B ) NRK-52E cells were exposed to HBSS with or without rhein 5 μg/ml for 6 hours and subjected to western blot analysis of p-p38. ( C ) NRK-52E cells were exposed to HBSS with or without SB203580 (a p38 inhibitor) 10 μM for 1 hour and subjected to western blot analysis of LC3 I/II and p-p38. ( D ) NRK-52E cells were exposed to HBSS with or without rhein 5 μg/ml for 6 hours and subjected to western blot analysis of p-Erk. ( E ) NRK-52E cells were exposed to HBSS with or without PD098059 (a p-Erk inhibitor) 50 μM for 1 hour and subjected to western blot analysis of LC3 I/II and p-Erk. ( F ) NRK-52E cells were transfected with mRFP-LC3 and treated with HBSS and bafilomycin A1 10 nM with or without SB203580 10 μM or PD098059 50 μM for 2 hours and subjected to fluorescence microscopy. Scale bar = 5 μm. Data are expressed as mean ± SD, * P

    Journal: Scientific Reports

    Article Title: Rhein Inhibits Autophagy in Rat Renal Tubular Cells by Regulation of AMPK/mTOR Signaling

    doi: 10.1038/srep43790

    Figure Lengend Snippet: Autophagic Activity is Inhibited by Rhein through the MAPKs Signaling Pathways. ( A ) NRK-52E cells were treated with HBSS for 0, 0.5, 1, 2 and 6 hours and subjected to western blot analysis of the phosphorylation of p38 (p-p38), Erk (p-Erk) and JNK (p-JNK). ( B ) NRK-52E cells were exposed to HBSS with or without rhein 5 μg/ml for 6 hours and subjected to western blot analysis of p-p38. ( C ) NRK-52E cells were exposed to HBSS with or without SB203580 (a p38 inhibitor) 10 μM for 1 hour and subjected to western blot analysis of LC3 I/II and p-p38. ( D ) NRK-52E cells were exposed to HBSS with or without rhein 5 μg/ml for 6 hours and subjected to western blot analysis of p-Erk. ( E ) NRK-52E cells were exposed to HBSS with or without PD098059 (a p-Erk inhibitor) 50 μM for 1 hour and subjected to western blot analysis of LC3 I/II and p-Erk. ( F ) NRK-52E cells were transfected with mRFP-LC3 and treated with HBSS and bafilomycin A1 10 nM with or without SB203580 10 μM or PD098059 50 μM for 2 hours and subjected to fluorescence microscopy. Scale bar = 5 μm. Data are expressed as mean ± SD, * P

    Article Snippet: Rhein, emodin, bafilomycin A1, LiCl, rapamycin, insulin, metformin, Akti, PD098059 and SB203580 were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO, USA).

    Techniques: Activity Assay, Western Blot, Transfection, Fluorescence, Microscopy

    Iron reduces TNF, but not IL1β, mRNA stability under hypoxia. ( A and B ) Caco-2 cells were subjected to hypoxia (0.2% O 2 ) for 24 hours in the presence or absence of 150 μmol/L DFO or 200 μmol/L FAC. Cells then were incubated with 5 μg/mL actinomycin D for 1 and 2 hours and the consequent decay of ( A ) TNF and ( B ) IL1β mRNA was monitored by quantitative PCR. Results represent means + SEM of 2 independent experiments performed in triplicate. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Iron Prevents Hypoxia-Associated Inflammation Through the Regulation of Nuclear Factor-κB in the Intestinal Epithelium

    doi: 10.1016/j.jcmgh.2018.10.006

    Figure Lengend Snippet: Iron reduces TNF, but not IL1β, mRNA stability under hypoxia. ( A and B ) Caco-2 cells were subjected to hypoxia (0.2% O 2 ) for 24 hours in the presence or absence of 150 μmol/L DFO or 200 μmol/L FAC. Cells then were incubated with 5 μg/mL actinomycin D for 1 and 2 hours and the consequent decay of ( A ) TNF and ( B ) IL1β mRNA was monitored by quantitative PCR. Results represent means + SEM of 2 independent experiments performed in triplicate. * P

    Article Snippet: In some experiments, Caco-2 cells were treated with 250 nmol/L rapamycin for 1 hour before hypoxia (Sigma-Aldrich). mRNA synthesis was inhibited using 5 μg/mL actinomycin D (Sigma-Aldrich).

    Techniques: Incubation, Real-time Polymerase Chain Reaction