rapamycin Search Results


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  • 99
    Thermo Fisher rapamycin
    miR‐199a‐3p targets the 3′ untranslated region (UTR) of MET and mammalian target of <t>rapamycin</t> (MTOR). 786‐O cells were co‐transfected for 24 h with a miR‐199a‐3p or non‐targeting
    Rapamycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rapamycin
    miR‐199a‐3p targets the 3′ untranslated region (UTR) of MET and mammalian target of <t>rapamycin</t> (MTOR). 786‐O cells were co‐transfected for 24 h with a miR‐199a‐3p or non‐targeting
    Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 13307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LC Laboratories sirolimus
    Effect of late initiation of <t>sirolimus</t> on renal p-S6 (A), p-4E-BP1 (B) and p-Akt. Representative photomicrographs of immunostaining for p-S6, p-4EBP1 and p-Akt in the kidney at week 20 from Lewis and LPK rats treated from week 10 until week 20. Scale bar = 100μm.
    Sirolimus, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc rapamycin
    Effect of late initiation of <t>sirolimus</t> on renal p-S6 (A), p-4E-BP1 (B) and p-Akt. Representative photomicrographs of immunostaining for p-S6, p-4EBP1 and p-Akt in the kidney at week 20 from Lewis and LPK rats treated from week 10 until week 20. Scale bar = 100μm.
    Rapamycin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1982 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals rapamycin
    <t>Rapamycin</t> promotes HEI-OC1 cell survival after cisplatin-induced damage. (A) The CCK8 assay was performed to examine cell viability of HEI-OC1 cells following CDDP (20 μM) exposure for 24 h with or without RA (0.5 μM; n = 3 individual experiments). (B) The image of HEI-OC1 cells following CDDP (20 μM) exposure for 24 h with or without RA (0.5 μM; n = 3 individual experiments). (C,D) Western blots and densitometry analysis for autophagy marker LC3-II and p62 in CDDP (20 μM) exposure for 24 h with or without RA (0.5 μM; n = 3). Data represent the mean ± SEM. * p
    Rapamycin, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 1078 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pfizer Inc sirolimus
    Effect of late initiation of <t>sirolimus</t> on renal p-S6 (A), p-4E-BP1 (B) and p-Akt. Representative photomicrographs of immunostaining for p-S6, p-4EBP1 and p-Akt in the kidney at week 20 from Lewis and LPK rats treated from week 10 until week 20. Scale bar = 100μm.
    Sirolimus, supplied by Pfizer Inc, used in various techniques. Bioz Stars score: 92/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Tocris rapamycin
    Effect of late initiation of <t>sirolimus</t> on renal p-S6 (A), p-4E-BP1 (B) and p-Akt. Representative photomicrographs of immunostaining for p-S6, p-4EBP1 and p-Akt in the kidney at week 20 from Lewis and LPK rats treated from week 10 until week 20. Scale bar = 100μm.
    Rapamycin, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 369 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cordis corporation sirolimus eluting stents
    Comparison of malapposition strut numbers according to stent. * Represents statistical significance ( p = 0.00), † indicates non-significant results ( p = 0.81). Definition of malapposition: ≥ 160 µm for SES, ≥ 130 µm for PES, ≥ 110 µm for ZES. 7 SES, <t>sirolimus-eluting</t> stent; PES, paclitaxel-eluting stent; ZES, zotarolimus-eluting stent.
    Sirolimus Eluting Stents, supplied by Cordis corporation, used in various techniques. Bioz Stars score: 89/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical rapamycin
    The deubiquitinase USP7 is a critical target of the mTOR pathway and reduces MCL-1 protein ubiquitination level, preventing MCL-1 proteasome degradation. A. Representative Western blot analysis of USP7 levels in BEAS-2B-Control, BEAS-2B-As, BEAS-2B-BaP and BEAS-2B-As+BaP cells. B. Representative Western blot analysis of USP7 protein levels in BEAS-2B-As+BaP cells pre-treated with different concentrations of Wortmannin or <t>rapamycin</t> for 24 h, followed by a vehicle control or 10 µM of MG132 co-treatment for 2 h. C. Representative Western blot analysis of USP7 and MCL-1 protein levels in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h. D. Representative Western blot analysis of MCL-1 IP experiment in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h. IP: immunoprecipitation; IB: immunoblotting; WCL: whole cell lysate. E. Representative Western blot analysis of MCL-1 protein levels in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h , followed by a vehicle control or 10 µM of MG132 co-treatment for 2 h. F. Representative Western blot analysis of total and cleaved PARP and caspase-3 protein levels in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h, followed by a vehicle control or 20 µM of ABT-737 co-treatment for 24 h. Similar results were obtained in the repeated experiments.
    Rapamycin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mtor inhibitor rapamycin
    Inhibition of <t>mTOR</t> signaling ameliorates ATF3 deficiency-mediated liver damage in IR-induced liver injury. Mice were injected with the mTOR inhibitor <t>rapamycin</t> (Rap) or DMSO vehicle at 60 min prior to ischemia. a Representative histological staining (H E, original magnification ×100) and TUNEL staining of ischemic liver tissue (4–6 mice/group). b Liver damage, as evaluated by Suzuki’s score. *** p
    Mtor Inhibitor Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 487 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Wyeth Biopharma rapamycin
    <t>Sirolimus</t> prevents weight gain and decreases fat mass of rats fed a HF diet
    Rapamycin, supplied by Wyeth Biopharma, used in various techniques. Bioz Stars score: 92/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam rapamycin
    <t>Sirolimus</t> prevents weight gain and decreases fat mass of rats fed a HF diet
    Rapamycin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 239 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co rapamycin
    PINK1 regulation by chloroquine and <t>rapamycin.</t> Identification of PINK1 ( a ) whole lysate from rat brain cells was used as positive control for protein identification. 15 μg of proteins from rat brain and human spermatozoa were loaded, separated by SDS-PAGE and immunoblotting was performed with a specific antibody against PINK1. PINK1, green signal, was studied by immunofluorescence, as described in material and methods in fresh samples ( b ). Regulation of PINK1 protein was studied in fresh and after 2 hours of incubation ( c ) and in the presence of chloroquine (50 μM) and rapamycin (100 nM) ( d ). Proteins were extracted, resolved and detected with specific antibody. Data from PINK1 was normalized respect to α-tubulin. Columns with asterisk indicate significant differences (P
    Rapamycin, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    InvivoGen rapamycin
    PINK1 regulation by chloroquine and <t>rapamycin.</t> Identification of PINK1 ( a ) whole lysate from rat brain cells was used as positive control for protein identification. 15 μg of proteins from rat brain and human spermatozoa were loaded, separated by SDS-PAGE and immunoblotting was performed with a specific antibody against PINK1. PINK1, green signal, was studied by immunofluorescence, as described in material and methods in fresh samples ( b ). Regulation of PINK1 protein was studied in fresh and after 2 hours of incubation ( c ) and in the presence of chloroquine (50 μM) and rapamycin (100 nM) ( d ). Proteins were extracted, resolved and detected with specific antibody. Data from PINK1 was normalized respect to α-tubulin. Columns with asterisk indicate significant differences (P
    Rapamycin, supplied by InvivoGen, used in various techniques. Bioz Stars score: 98/100, based on 218 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA rapamycin
    The RLR-mediated production of IFN-α and pro-inflammatory cytokines is decreased upon mTOR blockade in moDCs. Immature moDCs were pre-treated with vehicle control, 100 nM <t>rapamycin</t> (RAPA) or 100 nM AZD8055 (AZD) for 2 h and then stimulated with 3p-hpRNA (0.5 μg/ml) (A) , polyI:C (1 μg/ml) (B) or VSV (MOI 1) (C) . (A–C) IFN-α, IL-6 and TNF protein levels were measured by ELISA 24 h after 3p-hpRNA and polyI:C activation as well as 18 h after VSV stimulation. Data are shown as mean ± SD from 5 to 8 independent experiments. * p
    Rapamycin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH rapamycin
    Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were transfected with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with <t>rapamycin</t> (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0
    Rapamycin, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Beyotime rapamycin
    mTORC1 downregulates PDGFRɑ/AKT pathway through inhibition of FOXO3a ( A ) Tsc2−/− or Tsc1−/− MEFs were infected with lentivirus harboring a vector encoding FOXO3aTM (LV-FOXO3aTM) or the empty vector (LV). ( B ) Tsc2+/+ or Tsc1+/+ MEFs were transfected with two independent siRNAs targeting FOXO3a or the control siRNAs (siNC) for 48 h. A and B. Cell lysates and RNA were subjected to immunoblotting (upper panels) and qRT-PCR (lower panels), respectively. ( C ) Schematic representation of the putative wild-type (WT) and mutated (mut 1 and mut 2 ) FOXO3a-binding sites in the promoter of mouse PDGFRɑ gene. ( D ) HEK293T cells were co-transfected with pPDGFRɑ-Luc reporter plasmid plus FOXO3aTM expression vector (pLVX-FOXO3aTM) or control vector (pLVX) and the internal control plasmid pRL-TK. ( E ) Tsc2−/− MEFs were co-transfected with pLVX-FOXO3aTM plus pPDGFRɑ-Luc, pPDGFRɑ mut1 -Luc, or pPDGFRɑ mut2 -Luc reporter plasmid and pRL-TK plasmid. D and E. Relative luciferase activity was examined 24 h after transfection. ( F ) Tsc2−/− MEFs were transduced with LV-FOXO3aTM or LV lentiviruses. ( G ) Tsc1+/+, Tsc1−/−, or <t>rapamycin-treated</t> (20 nM 24 h) Tsc1−/− MEFs. F and G. Cells were subjected to ChIP assay using an anti-FOXO3a antibody. Normal rabbit IgG antibody served as the negative control. PCR was performed to amplify regions surrounding the putative FOXO3a-binding regions (PBR1 and PBR2) and a nonspecific FOXO3a-binding region (NBR). Representative data from two independent experiments were shown. ** P
    Rapamycin, supplied by Beyotime, used in various techniques. Bioz Stars score: 96/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Novartis rapamycin
    <t>Rapamycin</t> (Rapa) and the combination of cyclosporin A (CsA) and Rapa changes β 1 and β 3 integrin expression on MLR-activated cells. Peripheral blood mononuclear cells were cultured for 6 days in the presence of CsA (200 ng per 1 mL medium), Rapa (20 ng per 1 mL medium), and the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR) and then lysed in RIPA buffer. Protein extracts (15 μ g) were digested with endo- β -N-acetylglucosaminidase H (Endo H) from Streptomyces plicatus , SDS-PAGE-separated on 10% gel under reducing conditions, electroblotted onto a PVDF membrane, and probed with specific primary antibodies: mouse monoclonal anti- β 1 (Chemicon, MAB2251, clone B3B11) and rabbit polyclonal anti- β 3 (Chemicon, AB1932). Antibody-bound integrins were visualized by chemiluminescence. GAPDH was the endogenous control. C: untreated cells.
    Rapamycin, supplied by Novartis, used in various techniques. Bioz Stars score: 92/100, based on 168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    Rapamycin d is intended for use as an internal standard for the quantification of rapamycin Item No 13346 by GC or LC MS Rapamycin is an allosteric inhibitor of the
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    N/A
    mTOR inhibitor with anti cancer activity It has anti angiogenic effects which may contribute to the antitumor activity of temsirolimus observed in breast cancer Temsirolimus shows synergistic in vivo anti
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    N/A
    Rapamycin Item No 13346 is an immunosuppressant that specifically interacts with the cytosolic FK binding protein 12 to form a complex which inhibits the mammalian target of rapamycin mTOR pathway
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    Image Search Results


    miR‐199a‐3p targets the 3′ untranslated region (UTR) of MET and mammalian target of rapamycin (MTOR). 786‐O cells were co‐transfected for 24 h with a miR‐199a‐3p or non‐targeting

    Journal: Molecular Oncology

    Article Title: An integrated genomic analysis of papillary renal cell carcinoma type 1 uncovers the role of focal adhesion and extracellular matrix pathways), An integrated genomic analysis of papillary renal cell carcinoma type 1 uncovers the role of focal adhesion and extracellular matrix pathways

    doi: 10.1016/j.molonc.2015.04.007

    Figure Lengend Snippet: miR‐199a‐3p targets the 3′ untranslated region (UTR) of MET and mammalian target of rapamycin (MTOR). 786‐O cells were co‐transfected for 24 h with a miR‐199a‐3p or non‐targeting

    Article Snippet: The expression level of target genes CAV2 , integrin beta 8 ( ITGB8 ), MET and mammalian target of rapamycin ( MTOR) was measured using Fast SYBR Green Master Mix (Applied Biosystems).

    Techniques: Transfection

    Effect of late initiation of sirolimus on renal p-S6 (A), p-4E-BP1 (B) and p-Akt. Representative photomicrographs of immunostaining for p-S6, p-4EBP1 and p-Akt in the kidney at week 20 from Lewis and LPK rats treated from week 10 until week 20. Scale bar = 100μm.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on renal p-S6 (A), p-4E-BP1 (B) and p-Akt. Representative photomicrographs of immunostaining for p-S6, p-4EBP1 and p-Akt in the kidney at week 20 from Lewis and LPK rats treated from week 10 until week 20. Scale bar = 100μm.

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques: Immunostaining

    Effect of late initiation of sirolimus on body growth and kidney enlargement in Study 3. A. Representative photomicrographs showing effects of sirolimus on body size; B. Time-course of body weight in the experimental groups from week 10 to week 20; C. Representative photomicrographs of kidney enlargement in LPK rats; D. Effect of sirolimus on the percentage two-kidney weight to body weight ratio at week 20; Data expressed as Mean±SE in panel B, *P

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on body growth and kidney enlargement in Study 3. A. Representative photomicrographs showing effects of sirolimus on body size; B. Time-course of body weight in the experimental groups from week 10 to week 20; C. Representative photomicrographs of kidney enlargement in LPK rats; D. Effect of sirolimus on the percentage two-kidney weight to body weight ratio at week 20; Data expressed as Mean±SE in panel B, *P

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques:

    Effect of late initiation of sirolimus on the progression renal dysfunction and proteinuria at baseline (week 10) and at the end of treatment (week 20) (Study 3). A. Serum creatinine; B. Endogenous creatinine clearance; C. Urinary protein to creatinine ratio; D. Serum cholesterol. Data are expressed as mean±SD; *P

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on the progression renal dysfunction and proteinuria at baseline (week 10) and at the end of treatment (week 20) (Study 3). A. Serum creatinine; B. Endogenous creatinine clearance; C. Urinary protein to creatinine ratio; D. Serum cholesterol. Data are expressed as mean±SD; *P

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques:

    Early initiation of sirolimus reduces the increase in total kidney volume in LPK rats. A and B. Representative MR images of the experimental groups at weeks 6 and 10. An in-plane resolution of approximately 0.28–0.39mm and an effective through-plane resolution of 0.8mm for both the coronal and axial image sets within acceptable scan times (coronal: 6min 44sec and axial: 5min 26sec) was achieved. Signal-to noise ratio was sufficient to enable accurate segmentation and volume calculation using 3D SLICER. Anatomical detail of the kidneys was well visualized. Images from the 3D FIESTA sequence are shown. C. Graph showing total kidney volume (TKV) in the experimental groups at weeks 4 and 10. The TKV increased by ~2.4 times in both the Lewis+vehicle and LPK+sirolimus groups from weeks 4 to 10. In contrast in the LPK+vehicle group increased by 11.6 times over the same period. Data expressed as mean±SD; At week 4, Lewis+vehicle: n = 1; LPK+vehicle:; LPK+sirolimus:; n = 3–4 per group) and at week 10, Lewis+vehicle n = 3; Lewis+sirolimus n = 3; LPK+vehicle n = 6; LPK+sirolimus.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Early initiation of sirolimus reduces the increase in total kidney volume in LPK rats. A and B. Representative MR images of the experimental groups at weeks 6 and 10. An in-plane resolution of approximately 0.28–0.39mm and an effective through-plane resolution of 0.8mm for both the coronal and axial image sets within acceptable scan times (coronal: 6min 44sec and axial: 5min 26sec) was achieved. Signal-to noise ratio was sufficient to enable accurate segmentation and volume calculation using 3D SLICER. Anatomical detail of the kidneys was well visualized. Images from the 3D FIESTA sequence are shown. C. Graph showing total kidney volume (TKV) in the experimental groups at weeks 4 and 10. The TKV increased by ~2.4 times in both the Lewis+vehicle and LPK+sirolimus groups from weeks 4 to 10. In contrast in the LPK+vehicle group increased by 11.6 times over the same period. Data expressed as mean±SD; At week 4, Lewis+vehicle: n = 1; LPK+vehicle:; LPK+sirolimus:; n = 3–4 per group) and at week 10, Lewis+vehicle n = 3; Lewis+sirolimus n = 3; LPK+vehicle n = 6; LPK+sirolimus.

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques: Sequencing

    Effects of early initiation of sirolimus on body weight and kidney enlargement (Study 2). Early commencement of sirolimus reduces kidney enlargement in LPK rats. A. Photomicrographs showing effects of sirolimus on body size in LPK rats after seven weeks of treatment; B. Time-course of body weight in the experimental groups from weeks 3 to 10; C. Photomicrographs showing effects of sirolimus on kidney size in LPK rats after seven weeks of treatment; D. Effect of sirolimus on kidney enlargement, as assessed by the percentage two-kidney weight to body weight ratio at week 10. In Panel B, data expressed as mean±SE; *P

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effects of early initiation of sirolimus on body weight and kidney enlargement (Study 2). Early commencement of sirolimus reduces kidney enlargement in LPK rats. A. Photomicrographs showing effects of sirolimus on body size in LPK rats after seven weeks of treatment; B. Time-course of body weight in the experimental groups from weeks 3 to 10; C. Photomicrographs showing effects of sirolimus on kidney size in LPK rats after seven weeks of treatment; D. Effect of sirolimus on kidney enlargement, as assessed by the percentage two-kidney weight to body weight ratio at week 10. In Panel B, data expressed as mean±SE; *P

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques:

    Effect of late initiation of sirolimus on NF-κB-dependent proinflammatory gene expression ( TNFα and CCL2 ) in Lewis and LPK rats at Week 20 in Study 3. The mRNA expression is shown as the target gene corrected for GAPDH, and expressed as a fold-change over Lewis+V. Data are expressed as mean±SD; *p

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on NF-κB-dependent proinflammatory gene expression ( TNFα and CCL2 ) in Lewis and LPK rats at Week 20 in Study 3. The mRNA expression is shown as the target gene corrected for GAPDH, and expressed as a fold-change over Lewis+V. Data are expressed as mean±SD; *p

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques: Expressing

    Effect of sirolimus on ultrastructural abnormalities in renal cilia in LPK rats. Shown are representative SEM images of renal epithelial cells highlighting cilia (arrows) from Lewis untreated control (A: age 10 weeks) and LPK treated with vehicle (B: age 20 weeks, Study 3) or sirolimus (C: age 20 weeks, Study 3). Scale bars in panels A to C are each 10 μ m with images all taken at the same magnification (x 2200).

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of sirolimus on ultrastructural abnormalities in renal cilia in LPK rats. Shown are representative SEM images of renal epithelial cells highlighting cilia (arrows) from Lewis untreated control (A: age 10 weeks) and LPK treated with vehicle (B: age 20 weeks, Study 3) or sirolimus (C: age 20 weeks, Study 3). Scale bars in panels A to C are each 10 μ m with images all taken at the same magnification (x 2200).

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques:

    Representative immunohistochemistry images for p-S6 and p-4EBP1 following early initiation of sirolimus. Shown are representative photomicrographs of kidney cortices from Lewis and LPK rats given sirolimus from week 3 until week 10. Scale bar = 100μm.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Representative immunohistochemistry images for p-S6 and p-4EBP1 following early initiation of sirolimus. Shown are representative photomicrographs of kidney cortices from Lewis and LPK rats given sirolimus from week 3 until week 10. Scale bar = 100μm.

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques: Immunohistochemistry

    Effect of late initiation of sirolimus on renal p-S6 (A), p-4E-BP1 (B) and p-Akt (C) as assessed by quantitative analysis of immunostaining. Data (mean±SD) are expressed as the fold-change over the average for vehicle-treated animals; *p

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on renal p-S6 (A), p-4E-BP1 (B) and p-Akt (C) as assessed by quantitative analysis of immunostaining. Data (mean±SD) are expressed as the fold-change over the average for vehicle-treated animals; *p

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques: Immunostaining

    Effect of sirolimus on cardiovascular disease in LPK rats. Shown are changes in the heart to body weight (HW:BW) ratio in Studies 1, 2 and 3 (Panels A, B and C respectively) and systolic tail arterial blood pressure in Study 3 (Panel D). Data are expressed as mean±SD; *P

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of sirolimus on cardiovascular disease in LPK rats. Shown are changes in the heart to body weight (HW:BW) ratio in Studies 1, 2 and 3 (Panels A, B and C respectively) and systolic tail arterial blood pressure in Study 3 (Panel D). Data are expressed as mean±SD; *P

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques:

    Effect of late initiation of sirolimus on renal histology at Week 20 in LPK and Lewis rats. Shown are representative whole-slide digital images of sections stained with Sirius-red from the experimental group. Sirolimus administration from weeks 10 to 20 reduced kidney size but did not alter the percentage cyst area and interstitial fibrosis in LPK rats.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on renal histology at Week 20 in LPK and Lewis rats. Shown are representative whole-slide digital images of sections stained with Sirius-red from the experimental group. Sirolimus administration from weeks 10 to 20 reduced kidney size but did not alter the percentage cyst area and interstitial fibrosis in LPK rats.

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques: Staining

    Effects of early initiation of sirolimus on NF-κB dependent proinflammatory gene expression ( TNFα and CCL2 ) in Study 2. The mRNA expression is shown as the target gene corrected for GAPDH, and expressed as a fold-change over Lewis+vehicle (V). Data are expressed as mean±SD; **p

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effects of early initiation of sirolimus on NF-κB dependent proinflammatory gene expression ( TNFα and CCL2 ) in Study 2. The mRNA expression is shown as the target gene corrected for GAPDH, and expressed as a fold-change over Lewis+vehicle (V). Data are expressed as mean±SD; **p

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques: Expressing

    Effect of early initiation of sirolimus on p-S6 (A), p-4E-BP1 (B) and p-Akt (C), as assessed by quantitative analysis of immunostaining. Data (mean±SD) are expressed as the fold-change over the average for vehicle-treated animals; *p

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of early initiation of sirolimus on p-S6 (A), p-4E-BP1 (B) and p-Akt (C), as assessed by quantitative analysis of immunostaining. Data (mean±SD) are expressed as the fold-change over the average for vehicle-treated animals; *p

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques: Immunostaining

    Effect of late initiation of sirolimus on the progression of total kidney volume in LPK rats (Study 3). A. Representative serial MRI scans of LPK rats treated with either vehicle (V) or sirolimus (S) from week 10 to 17. Treatment with sirolimus prevented the increase in total kidney volume (TKV); B. Quantitative analysis of TKV in the experimental groups. The increase in TKV between weeks 10 and 17 was 1.9 times in LPK+V, compared to 1.1 times in the LPK+S group; n = 4 per group per timepoint.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on the progression of total kidney volume in LPK rats (Study 3). A. Representative serial MRI scans of LPK rats treated with either vehicle (V) or sirolimus (S) from week 10 to 17. Treatment with sirolimus prevented the increase in total kidney volume (TKV); B. Quantitative analysis of TKV in the experimental groups. The increase in TKV between weeks 10 and 17 was 1.9 times in LPK+V, compared to 1.1 times in the LPK+S group; n = 4 per group per timepoint.

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques: Magnetic Resonance Imaging

    Effect of late initiation of sirolimus on cardiac histology at week 20. Shown are representative photomicrographs of heart tissue for interstitial collagen deposition (Sirius Red, upper panels) and p-S6 (lower panels) in Lewis and LPK rats at week 20 (Study 3). Scale bar = 100μm.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on cardiac histology at week 20. Shown are representative photomicrographs of heart tissue for interstitial collagen deposition (Sirius Red, upper panels) and p-S6 (lower panels) in Lewis and LPK rats at week 20 (Study 3). Scale bar = 100μm.

    Article Snippet: In the second pilot, a dose-finding study showed subcutaneous injections of sirolimus (obtained from LC Laboratories) at 0.2 mg/kg/day was well tolerated and reduced kidney enlargement over 1 week (data not shown).

    Techniques:

    Rapamycin promotes HEI-OC1 cell survival after cisplatin-induced damage. (A) The CCK8 assay was performed to examine cell viability of HEI-OC1 cells following CDDP (20 μM) exposure for 24 h with or without RA (0.5 μM; n = 3 individual experiments). (B) The image of HEI-OC1 cells following CDDP (20 μM) exposure for 24 h with or without RA (0.5 μM; n = 3 individual experiments). (C,D) Western blots and densitometry analysis for autophagy marker LC3-II and p62 in CDDP (20 μM) exposure for 24 h with or without RA (0.5 μM; n = 3). Data represent the mean ± SEM. * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Sirtuin 1 and Autophagy Attenuate Cisplatin-Induced Hair Cell Death in the Mouse Cochlea and Zebrafish Lateral Line

    doi: 10.3389/fncel.2018.00515

    Figure Lengend Snippet: Rapamycin promotes HEI-OC1 cell survival after cisplatin-induced damage. (A) The CCK8 assay was performed to examine cell viability of HEI-OC1 cells following CDDP (20 μM) exposure for 24 h with or without RA (0.5 μM; n = 3 individual experiments). (B) The image of HEI-OC1 cells following CDDP (20 μM) exposure for 24 h with or without RA (0.5 μM; n = 3 individual experiments). (C,D) Western blots and densitometry analysis for autophagy marker LC3-II and p62 in CDDP (20 μM) exposure for 24 h with or without RA (0.5 μM; n = 3). Data represent the mean ± SEM. * p

    Article Snippet: Rapamycin (RA, Selleck, S1039, TX, USA) was dissolved in DMSO, and intragastric administration (7.5 mg/kg) was performed three times 24 h before and after CDDP exposure, and 1 h before CDDP exposure.

    Techniques: CCK-8 Assay, Western Blot, Marker

    Effect of late initiation of sirolimus on renal p-S6 (A), p-4E-BP1 (B) and p-Akt. Representative photomicrographs of immunostaining for p-S6, p-4EBP1 and p-Akt in the kidney at week 20 from Lewis and LPK rats treated from week 10 until week 20. Scale bar = 100μm.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on renal p-S6 (A), p-4E-BP1 (B) and p-Akt. Representative photomicrographs of immunostaining for p-S6, p-4EBP1 and p-Akt in the kidney at week 20 from Lewis and LPK rats treated from week 10 until week 20. Scale bar = 100μm.

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques: Immunostaining

    Effect of late initiation of sirolimus on body growth and kidney enlargement in Study 3. A. Representative photomicrographs showing effects of sirolimus on body size; B. Time-course of body weight in the experimental groups from week 10 to week 20; C. Representative photomicrographs of kidney enlargement in LPK rats; D. Effect of sirolimus on the percentage two-kidney weight to body weight ratio at week 20; Data expressed as Mean±SE in panel B, *P

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on body growth and kidney enlargement in Study 3. A. Representative photomicrographs showing effects of sirolimus on body size; B. Time-course of body weight in the experimental groups from week 10 to week 20; C. Representative photomicrographs of kidney enlargement in LPK rats; D. Effect of sirolimus on the percentage two-kidney weight to body weight ratio at week 20; Data expressed as Mean±SE in panel B, *P

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques:

    Effect of late initiation of sirolimus on the progression renal dysfunction and proteinuria at baseline (week 10) and at the end of treatment (week 20) (Study 3). A. Serum creatinine; B. Endogenous creatinine clearance; C. Urinary protein to creatinine ratio; D. Serum cholesterol. Data are expressed as mean±SD; *P

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on the progression renal dysfunction and proteinuria at baseline (week 10) and at the end of treatment (week 20) (Study 3). A. Serum creatinine; B. Endogenous creatinine clearance; C. Urinary protein to creatinine ratio; D. Serum cholesterol. Data are expressed as mean±SD; *P

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques:

    Early initiation of sirolimus reduces the increase in total kidney volume in LPK rats. A and B. Representative MR images of the experimental groups at weeks 6 and 10. An in-plane resolution of approximately 0.28–0.39mm and an effective through-plane resolution of 0.8mm for both the coronal and axial image sets within acceptable scan times (coronal: 6min 44sec and axial: 5min 26sec) was achieved. Signal-to noise ratio was sufficient to enable accurate segmentation and volume calculation using 3D SLICER. Anatomical detail of the kidneys was well visualized. Images from the 3D FIESTA sequence are shown. C. Graph showing total kidney volume (TKV) in the experimental groups at weeks 4 and 10. The TKV increased by ~2.4 times in both the Lewis+vehicle and LPK+sirolimus groups from weeks 4 to 10. In contrast in the LPK+vehicle group increased by 11.6 times over the same period. Data expressed as mean±SD; At week 4, Lewis+vehicle: n = 1; LPK+vehicle:; LPK+sirolimus:; n = 3–4 per group) and at week 10, Lewis+vehicle n = 3; Lewis+sirolimus n = 3; LPK+vehicle n = 6; LPK+sirolimus.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Early initiation of sirolimus reduces the increase in total kidney volume in LPK rats. A and B. Representative MR images of the experimental groups at weeks 6 and 10. An in-plane resolution of approximately 0.28–0.39mm and an effective through-plane resolution of 0.8mm for both the coronal and axial image sets within acceptable scan times (coronal: 6min 44sec and axial: 5min 26sec) was achieved. Signal-to noise ratio was sufficient to enable accurate segmentation and volume calculation using 3D SLICER. Anatomical detail of the kidneys was well visualized. Images from the 3D FIESTA sequence are shown. C. Graph showing total kidney volume (TKV) in the experimental groups at weeks 4 and 10. The TKV increased by ~2.4 times in both the Lewis+vehicle and LPK+sirolimus groups from weeks 4 to 10. In contrast in the LPK+vehicle group increased by 11.6 times over the same period. Data expressed as mean±SD; At week 4, Lewis+vehicle: n = 1; LPK+vehicle:; LPK+sirolimus:; n = 3–4 per group) and at week 10, Lewis+vehicle n = 3; Lewis+sirolimus n = 3; LPK+vehicle n = 6; LPK+sirolimus.

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques: Sequencing

    Effects of early initiation of sirolimus on body weight and kidney enlargement (Study 2). Early commencement of sirolimus reduces kidney enlargement in LPK rats. A. Photomicrographs showing effects of sirolimus on body size in LPK rats after seven weeks of treatment; B. Time-course of body weight in the experimental groups from weeks 3 to 10; C. Photomicrographs showing effects of sirolimus on kidney size in LPK rats after seven weeks of treatment; D. Effect of sirolimus on kidney enlargement, as assessed by the percentage two-kidney weight to body weight ratio at week 10. In Panel B, data expressed as mean±SE; *P

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effects of early initiation of sirolimus on body weight and kidney enlargement (Study 2). Early commencement of sirolimus reduces kidney enlargement in LPK rats. A. Photomicrographs showing effects of sirolimus on body size in LPK rats after seven weeks of treatment; B. Time-course of body weight in the experimental groups from weeks 3 to 10; C. Photomicrographs showing effects of sirolimus on kidney size in LPK rats after seven weeks of treatment; D. Effect of sirolimus on kidney enlargement, as assessed by the percentage two-kidney weight to body weight ratio at week 10. In Panel B, data expressed as mean±SE; *P

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques:

    Effect of late initiation of sirolimus on NF-κB-dependent proinflammatory gene expression ( TNFα and CCL2 ) in Lewis and LPK rats at Week 20 in Study 3. The mRNA expression is shown as the target gene corrected for GAPDH, and expressed as a fold-change over Lewis+V. Data are expressed as mean±SD; *p

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on NF-κB-dependent proinflammatory gene expression ( TNFα and CCL2 ) in Lewis and LPK rats at Week 20 in Study 3. The mRNA expression is shown as the target gene corrected for GAPDH, and expressed as a fold-change over Lewis+V. Data are expressed as mean±SD; *p

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques: Expressing

    Effect of sirolimus on ultrastructural abnormalities in renal cilia in LPK rats. Shown are representative SEM images of renal epithelial cells highlighting cilia (arrows) from Lewis untreated control (A: age 10 weeks) and LPK treated with vehicle (B: age 20 weeks, Study 3) or sirolimus (C: age 20 weeks, Study 3). Scale bars in panels A to C are each 10 μ m with images all taken at the same magnification (x 2200).

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of sirolimus on ultrastructural abnormalities in renal cilia in LPK rats. Shown are representative SEM images of renal epithelial cells highlighting cilia (arrows) from Lewis untreated control (A: age 10 weeks) and LPK treated with vehicle (B: age 20 weeks, Study 3) or sirolimus (C: age 20 weeks, Study 3). Scale bars in panels A to C are each 10 μ m with images all taken at the same magnification (x 2200).

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques:

    Representative immunohistochemistry images for p-S6 and p-4EBP1 following early initiation of sirolimus. Shown are representative photomicrographs of kidney cortices from Lewis and LPK rats given sirolimus from week 3 until week 10. Scale bar = 100μm.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Representative immunohistochemistry images for p-S6 and p-4EBP1 following early initiation of sirolimus. Shown are representative photomicrographs of kidney cortices from Lewis and LPK rats given sirolimus from week 3 until week 10. Scale bar = 100μm.

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques: Immunohistochemistry

    Effect of late initiation of sirolimus on renal p-S6 (A), p-4E-BP1 (B) and p-Akt (C) as assessed by quantitative analysis of immunostaining. Data (mean±SD) are expressed as the fold-change over the average for vehicle-treated animals; *p

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on renal p-S6 (A), p-4E-BP1 (B) and p-Akt (C) as assessed by quantitative analysis of immunostaining. Data (mean±SD) are expressed as the fold-change over the average for vehicle-treated animals; *p

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques: Immunostaining

    Effect of sirolimus on cardiovascular disease in LPK rats. Shown are changes in the heart to body weight (HW:BW) ratio in Studies 1, 2 and 3 (Panels A, B and C respectively) and systolic tail arterial blood pressure in Study 3 (Panel D). Data are expressed as mean±SD; *P

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of sirolimus on cardiovascular disease in LPK rats. Shown are changes in the heart to body weight (HW:BW) ratio in Studies 1, 2 and 3 (Panels A, B and C respectively) and systolic tail arterial blood pressure in Study 3 (Panel D). Data are expressed as mean±SD; *P

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques:

    Effect of late initiation of sirolimus on renal histology at Week 20 in LPK and Lewis rats. Shown are representative whole-slide digital images of sections stained with Sirius-red from the experimental group. Sirolimus administration from weeks 10 to 20 reduced kidney size but did not alter the percentage cyst area and interstitial fibrosis in LPK rats.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on renal histology at Week 20 in LPK and Lewis rats. Shown are representative whole-slide digital images of sections stained with Sirius-red from the experimental group. Sirolimus administration from weeks 10 to 20 reduced kidney size but did not alter the percentage cyst area and interstitial fibrosis in LPK rats.

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques: Staining

    Effects of early initiation of sirolimus on NF-κB dependent proinflammatory gene expression ( TNFα and CCL2 ) in Study 2. The mRNA expression is shown as the target gene corrected for GAPDH, and expressed as a fold-change over Lewis+vehicle (V). Data are expressed as mean±SD; **p

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effects of early initiation of sirolimus on NF-κB dependent proinflammatory gene expression ( TNFα and CCL2 ) in Study 2. The mRNA expression is shown as the target gene corrected for GAPDH, and expressed as a fold-change over Lewis+vehicle (V). Data are expressed as mean±SD; **p

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques: Expressing

    Effect of early initiation of sirolimus on p-S6 (A), p-4E-BP1 (B) and p-Akt (C), as assessed by quantitative analysis of immunostaining. Data (mean±SD) are expressed as the fold-change over the average for vehicle-treated animals; *p

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of early initiation of sirolimus on p-S6 (A), p-4E-BP1 (B) and p-Akt (C), as assessed by quantitative analysis of immunostaining. Data (mean±SD) are expressed as the fold-change over the average for vehicle-treated animals; *p

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques: Immunostaining

    Effect of late initiation of sirolimus on the progression of total kidney volume in LPK rats (Study 3). A. Representative serial MRI scans of LPK rats treated with either vehicle (V) or sirolimus (S) from week 10 to 17. Treatment with sirolimus prevented the increase in total kidney volume (TKV); B. Quantitative analysis of TKV in the experimental groups. The increase in TKV between weeks 10 and 17 was 1.9 times in LPK+V, compared to 1.1 times in the LPK+S group; n = 4 per group per timepoint.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on the progression of total kidney volume in LPK rats (Study 3). A. Representative serial MRI scans of LPK rats treated with either vehicle (V) or sirolimus (S) from week 10 to 17. Treatment with sirolimus prevented the increase in total kidney volume (TKV); B. Quantitative analysis of TKV in the experimental groups. The increase in TKV between weeks 10 and 17 was 1.9 times in LPK+V, compared to 1.1 times in the LPK+S group; n = 4 per group per timepoint.

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques: Magnetic Resonance Imaging

    Effect of late initiation of sirolimus on cardiac histology at week 20. Shown are representative photomicrographs of heart tissue for interstitial collagen deposition (Sirius Red, upper panels) and p-S6 (lower panels) in Lewis and LPK rats at week 20 (Study 3). Scale bar = 100μm.

    Journal: PLoS ONE

    Article Title: Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

    doi: 10.1371/journal.pone.0164193

    Figure Lengend Snippet: Effect of late initiation of sirolimus on cardiac histology at week 20. Shown are representative photomicrographs of heart tissue for interstitial collagen deposition (Sirius Red, upper panels) and p-S6 (lower panels) in Lewis and LPK rats at week 20 (Study 3). Scale bar = 100μm.

    Article Snippet: Briefly, in the first pilot, treatment with sirolimus in drinking water (Rapamune, Pfizer; 1mg/ml; 2 mg/kg/day) for 8 weeks in LPK rats only partly attenuated kidney enlargement and whole blood levels of sirolimus were variable.

    Techniques:

    Comparison of malapposition strut numbers according to stent. * Represents statistical significance ( p = 0.00), † indicates non-significant results ( p = 0.81). Definition of malapposition: ≥ 160 µm for SES, ≥ 130 µm for PES, ≥ 110 µm for ZES. 7 SES, sirolimus-eluting stent; PES, paclitaxel-eluting stent; ZES, zotarolimus-eluting stent.

    Journal: Yonsei Medical Journal

    Article Title: The Initial Extent of Malapposition in ST-Elevation Myocardial Infarction Treated with Drug-Eluting Stent: The Usefulness of Optical Coherence Tomography

    doi: 10.3349/ymj.2010.51.3.332

    Figure Lengend Snippet: Comparison of malapposition strut numbers according to stent. * Represents statistical significance ( p = 0.00), † indicates non-significant results ( p = 0.81). Definition of malapposition: ≥ 160 µm for SES, ≥ 130 µm for PES, ≥ 110 µm for ZES. 7 SES, sirolimus-eluting stent; PES, paclitaxel-eluting stent; ZES, zotarolimus-eluting stent.

    Article Snippet: Malapposed stent struts were defined as struts with detachment from the vessel wall ≥ 160 µm for sirolimus-eluting stents (SES; Cypher select, Cordis, J & J, East Bridgewater, NJ, USA), ≥ 130 µm for paclitaxeleluting stents (PES; Taxus Liberte, Boston Scientific, Natick, MA, USA), and ≥ 110 µm for zotaroliums-eluting stents (ZES; Endeavor, Medtronic, Minneapolis, MN, USA).

    Techniques:

    Average distances between lumen and stent. * Represents statistical significance ( p = 0.00). Definition of incomplete stent apposition: ≥ 160 µm for SES, ≥ 130 µm for PES, ≥ 110 µm for ZES. SES, sirolimus-eluting stent; PES, paclitaxel-eluting stent; ZES, zotarolimus-eluting stent. 7

    Journal: Yonsei Medical Journal

    Article Title: The Initial Extent of Malapposition in ST-Elevation Myocardial Infarction Treated with Drug-Eluting Stent: The Usefulness of Optical Coherence Tomography

    doi: 10.3349/ymj.2010.51.3.332

    Figure Lengend Snippet: Average distances between lumen and stent. * Represents statistical significance ( p = 0.00). Definition of incomplete stent apposition: ≥ 160 µm for SES, ≥ 130 µm for PES, ≥ 110 µm for ZES. SES, sirolimus-eluting stent; PES, paclitaxel-eluting stent; ZES, zotarolimus-eluting stent. 7

    Article Snippet: Malapposed stent struts were defined as struts with detachment from the vessel wall ≥ 160 µm for sirolimus-eluting stents (SES; Cypher select, Cordis, J & J, East Bridgewater, NJ, USA), ≥ 130 µm for paclitaxeleluting stents (PES; Taxus Liberte, Boston Scientific, Natick, MA, USA), and ≥ 110 µm for zotaroliums-eluting stents (ZES; Endeavor, Medtronic, Minneapolis, MN, USA).

    Techniques:

    Measurements of strut apposition. The distances between the endo-luminal surface of the strut reflection and the vessel wall were measured. (A and B) shows OCT image of SES and strut measurements and represents malapposition. (C and D) shows OCT image of PES and strut measurement shows normal stent apposition. (E and F) represents OCT image of ZES and its measurement shows normal stent apposition. Definition of malapposition: ≥ 160 µm for SES, ≥ 130 µm for PES, ≥ 110 µm for ZES. 7 OCT, optical coherence tomography; SES, sirolimus-eluting stent; PES, paclitaxel-eluting stent; ZES, zotarolimus-eluting stent.

    Journal: Yonsei Medical Journal

    Article Title: The Initial Extent of Malapposition in ST-Elevation Myocardial Infarction Treated with Drug-Eluting Stent: The Usefulness of Optical Coherence Tomography

    doi: 10.3349/ymj.2010.51.3.332

    Figure Lengend Snippet: Measurements of strut apposition. The distances between the endo-luminal surface of the strut reflection and the vessel wall were measured. (A and B) shows OCT image of SES and strut measurements and represents malapposition. (C and D) shows OCT image of PES and strut measurement shows normal stent apposition. (E and F) represents OCT image of ZES and its measurement shows normal stent apposition. Definition of malapposition: ≥ 160 µm for SES, ≥ 130 µm for PES, ≥ 110 µm for ZES. 7 OCT, optical coherence tomography; SES, sirolimus-eluting stent; PES, paclitaxel-eluting stent; ZES, zotarolimus-eluting stent.

    Article Snippet: Malapposed stent struts were defined as struts with detachment from the vessel wall ≥ 160 µm for sirolimus-eluting stents (SES; Cypher select, Cordis, J & J, East Bridgewater, NJ, USA), ≥ 130 µm for paclitaxeleluting stents (PES; Taxus Liberte, Boston Scientific, Natick, MA, USA), and ≥ 110 µm for zotaroliums-eluting stents (ZES; Endeavor, Medtronic, Minneapolis, MN, USA).

    Techniques:

    The deubiquitinase USP7 is a critical target of the mTOR pathway and reduces MCL-1 protein ubiquitination level, preventing MCL-1 proteasome degradation. A. Representative Western blot analysis of USP7 levels in BEAS-2B-Control, BEAS-2B-As, BEAS-2B-BaP and BEAS-2B-As+BaP cells. B. Representative Western blot analysis of USP7 protein levels in BEAS-2B-As+BaP cells pre-treated with different concentrations of Wortmannin or rapamycin for 24 h, followed by a vehicle control or 10 µM of MG132 co-treatment for 2 h. C. Representative Western blot analysis of USP7 and MCL-1 protein levels in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h. D. Representative Western blot analysis of MCL-1 IP experiment in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h. IP: immunoprecipitation; IB: immunoblotting; WCL: whole cell lysate. E. Representative Western blot analysis of MCL-1 protein levels in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h , followed by a vehicle control or 10 µM of MG132 co-treatment for 2 h. F. Representative Western blot analysis of total and cleaved PARP and caspase-3 protein levels in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h, followed by a vehicle control or 20 µM of ABT-737 co-treatment for 24 h. Similar results were obtained in the repeated experiments.

    Journal: Theranostics

    Article Title: Deubiquitinase USP7-mediated MCL-1 up-regulation enhances Arsenic and Benzo(a)pyrene co-exposure-induced Cancer Stem Cell-like property and Tumorigenesis

    doi: 10.7150/thno.47897

    Figure Lengend Snippet: The deubiquitinase USP7 is a critical target of the mTOR pathway and reduces MCL-1 protein ubiquitination level, preventing MCL-1 proteasome degradation. A. Representative Western blot analysis of USP7 levels in BEAS-2B-Control, BEAS-2B-As, BEAS-2B-BaP and BEAS-2B-As+BaP cells. B. Representative Western blot analysis of USP7 protein levels in BEAS-2B-As+BaP cells pre-treated with different concentrations of Wortmannin or rapamycin for 24 h, followed by a vehicle control or 10 µM of MG132 co-treatment for 2 h. C. Representative Western blot analysis of USP7 and MCL-1 protein levels in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h. D. Representative Western blot analysis of MCL-1 IP experiment in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h. IP: immunoprecipitation; IB: immunoblotting; WCL: whole cell lysate. E. Representative Western blot analysis of MCL-1 protein levels in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h , followed by a vehicle control or 10 µM of MG132 co-treatment for 2 h. F. Representative Western blot analysis of total and cleaved PARP and caspase-3 protein levels in BEAS-2B-As+BaP cells transfected with control siRNA (siControl) or USP7 siRNA (siUSP7) oligoes for 48 h, followed by a vehicle control or 20 µM of ABT-737 co-treatment for 24 h. Similar results were obtained in the repeated experiments.

    Article Snippet: ABT-737 (#11501), wortmannin (#10010591), rapamycin (#13346), cycloheximide (Chx) (#14126), and MG132 (#10012628) were purchased from Cayman Chemical company (Cayman Chemical, MI, USA).

    Techniques: Western Blot, Transfection, Immunoprecipitation

    Inhibition of the mTOR pathway significantly reduces MCL-1 protein stability in arsenic and BaP co-exposure-transformed cells and reverses their apoptosis resistance. A. Representative Western blot analysis of the mTOR pathway protein phosphorylation levels in the BEAS-2B-Control, BEAS-2B-As, BEAS-2B-BaP and BEAS-2B-As+BaP cells. B. Representative western blot analysis of the effects of different concentrations of Wortmannin (0.25, 0.5, 1.0, 2.0 µM) or rapamycin (0.03, 0.1, 0.3, 1.0 nM) treatment (24 h) on the Akt/mTOR pathway protein phosphorylation levels in BEAS-2B-As+BaP cells. C. Representative Western blot analysis and the quantitated results (mean ± SD, n=3) of MCL-1 protein half-life in BEAS-2B-As+BaP cells treated with 5 µM of cycloheximide (Chx) for 0, 20, 40, 60, or 80 minutes with or without rapamycin pre-treatment (1 nM, 2 h). * p

    Journal: Theranostics

    Article Title: Deubiquitinase USP7-mediated MCL-1 up-regulation enhances Arsenic and Benzo(a)pyrene co-exposure-induced Cancer Stem Cell-like property and Tumorigenesis

    doi: 10.7150/thno.47897

    Figure Lengend Snippet: Inhibition of the mTOR pathway significantly reduces MCL-1 protein stability in arsenic and BaP co-exposure-transformed cells and reverses their apoptosis resistance. A. Representative Western blot analysis of the mTOR pathway protein phosphorylation levels in the BEAS-2B-Control, BEAS-2B-As, BEAS-2B-BaP and BEAS-2B-As+BaP cells. B. Representative western blot analysis of the effects of different concentrations of Wortmannin (0.25, 0.5, 1.0, 2.0 µM) or rapamycin (0.03, 0.1, 0.3, 1.0 nM) treatment (24 h) on the Akt/mTOR pathway protein phosphorylation levels in BEAS-2B-As+BaP cells. C. Representative Western blot analysis and the quantitated results (mean ± SD, n=3) of MCL-1 protein half-life in BEAS-2B-As+BaP cells treated with 5 µM of cycloheximide (Chx) for 0, 20, 40, 60, or 80 minutes with or without rapamycin pre-treatment (1 nM, 2 h). * p

    Article Snippet: ABT-737 (#11501), wortmannin (#10010591), rapamycin (#13346), cycloheximide (Chx) (#14126), and MG132 (#10012628) were purchased from Cayman Chemical company (Cayman Chemical, MI, USA).

    Techniques: Inhibition, Transformation Assay, Western Blot

    Stably expressing MCL-1 prevents Wortmannin or rapamycin plus ABT-737 treatment from inducing apoptosis in arsenic and BaP co-exposure-transformed cells. A. Representative Western blot analysis of MCL-1, BCL-XL, BCL-2, Puma, Bax and Bim protein levels in vector control and MCL-1 stably overexpressing BEAS-2B-As+BaP cells. B. Representative Western blot analysis of total and cleaved PARP and caspase-3 protein levels in vector control and MCL-1 stably overexpressing BEAS-2B-As+BaP cells treated with a vehicle control, Wortmannin (2 µM), or rapamycin (1 nM) plus 20 µM of ABT-737 for 24 h. Similar results were obtained in the repeated experiments.

    Journal: Theranostics

    Article Title: Deubiquitinase USP7-mediated MCL-1 up-regulation enhances Arsenic and Benzo(a)pyrene co-exposure-induced Cancer Stem Cell-like property and Tumorigenesis

    doi: 10.7150/thno.47897

    Figure Lengend Snippet: Stably expressing MCL-1 prevents Wortmannin or rapamycin plus ABT-737 treatment from inducing apoptosis in arsenic and BaP co-exposure-transformed cells. A. Representative Western blot analysis of MCL-1, BCL-XL, BCL-2, Puma, Bax and Bim protein levels in vector control and MCL-1 stably overexpressing BEAS-2B-As+BaP cells. B. Representative Western blot analysis of total and cleaved PARP and caspase-3 protein levels in vector control and MCL-1 stably overexpressing BEAS-2B-As+BaP cells treated with a vehicle control, Wortmannin (2 µM), or rapamycin (1 nM) plus 20 µM of ABT-737 for 24 h. Similar results were obtained in the repeated experiments.

    Article Snippet: ABT-737 (#11501), wortmannin (#10010591), rapamycin (#13346), cycloheximide (Chx) (#14126), and MG132 (#10012628) were purchased from Cayman Chemical company (Cayman Chemical, MI, USA).

    Techniques: Stable Transfection, Expressing, Transformation Assay, Western Blot, Plasmid Preparation

    Inhibition of mTOR signaling ameliorates ATF3 deficiency-mediated liver damage in IR-induced liver injury. Mice were injected with the mTOR inhibitor rapamycin (Rap) or DMSO vehicle at 60 min prior to ischemia. a Representative histological staining (H E, original magnification ×100) and TUNEL staining of ischemic liver tissue (4–6 mice/group). b Liver damage, as evaluated by Suzuki’s score. *** p

    Journal: Cell Death & Disease

    Article Title: Loss of ATF3 exacerbates liver damage through the activation of mTOR/p70S6K/ HIF-1α signaling pathway in liver inflammatory injury

    doi: 10.1038/s41419-018-0894-1

    Figure Lengend Snippet: Inhibition of mTOR signaling ameliorates ATF3 deficiency-mediated liver damage in IR-induced liver injury. Mice were injected with the mTOR inhibitor rapamycin (Rap) or DMSO vehicle at 60 min prior to ischemia. a Representative histological staining (H E, original magnification ×100) and TUNEL staining of ischemic liver tissue (4–6 mice/group). b Liver damage, as evaluated by Suzuki’s score. *** p

    Article Snippet: Mice were injected with mTOR inhibitor Rapamycin (5 mg/kg, i.p. Calbiochem, Burlington, MA) or DMSO vehicle at 60 min prior to ischemia.

    Techniques: Inhibition, Mouse Assay, Injection, Staining, TUNEL Assay

    Sirolimus prevents weight gain and decreases fat mass of rats fed a HF diet

    Journal: British Journal of Pharmacology

    Article Title: Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

    doi: 10.1111/j.1476-5381.2011.01716.x

    Figure Lengend Snippet: Sirolimus prevents weight gain and decreases fat mass of rats fed a HF diet

    Article Snippet: Sirolimus is a clinically formulated injectable form of rapamycin, which contains, in addition to rapamycin, other inactive components (0.1% sodium CMC, 0.25% Polysobate 80) and was kindly provided by Wyeth Pharma GmbH (Munster, Germany).

    Techniques:

    Sirolimus prevents triglyceride accumulation and PTEN down-regulation in the liver of HF-fed rats. Wistar rats were fed a HF diet for 6 weeks. During the last 3 weeks of the experiment, HF fed animals were divided into two groups either treated with 2

    Journal: British Journal of Pharmacology

    Article Title: Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

    doi: 10.1111/j.1476-5381.2011.01716.x

    Figure Lengend Snippet: Sirolimus prevents triglyceride accumulation and PTEN down-regulation in the liver of HF-fed rats. Wistar rats were fed a HF diet for 6 weeks. During the last 3 weeks of the experiment, HF fed animals were divided into two groups either treated with 2

    Article Snippet: Sirolimus is a clinically formulated injectable form of rapamycin, which contains, in addition to rapamycin, other inactive components (0.1% sodium CMC, 0.25% Polysobate 80) and was kindly provided by Wyeth Pharma GmbH (Munster, Germany).

    Techniques:

    Effect of chronic Sirolimus administration on body weight (BW) gain and composition of Wistar rats fed a high fat (HF) diet. Wistar rats were fed a HF diet for 6 weeks. The last 3 weeks of the experiment, HF diet animals were chronically administered

    Journal: British Journal of Pharmacology

    Article Title: Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

    doi: 10.1111/j.1476-5381.2011.01716.x

    Figure Lengend Snippet: Effect of chronic Sirolimus administration on body weight (BW) gain and composition of Wistar rats fed a high fat (HF) diet. Wistar rats were fed a HF diet for 6 weeks. The last 3 weeks of the experiment, HF diet animals were chronically administered

    Article Snippet: Sirolimus is a clinically formulated injectable form of rapamycin, which contains, in addition to rapamycin, other inactive components (0.1% sodium CMC, 0.25% Polysobate 80) and was kindly provided by Wyeth Pharma GmbH (Munster, Germany).

    Techniques:

    Sirolimus prevents hepatic steatosis but exacerbates glucose intolerance and insulin resistance in rats fed a HF diet

    Journal: British Journal of Pharmacology

    Article Title: Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

    doi: 10.1111/j.1476-5381.2011.01716.x

    Figure Lengend Snippet: Sirolimus prevents hepatic steatosis but exacerbates glucose intolerance and insulin resistance in rats fed a HF diet

    Article Snippet: Sirolimus is a clinically formulated injectable form of rapamycin, which contains, in addition to rapamycin, other inactive components (0.1% sodium CMC, 0.25% Polysobate 80) and was kindly provided by Wyeth Pharma GmbH (Munster, Germany).

    Techniques:

    Chronic Sirolimus administration exacerbates glucose intolerance of rats fed a high-fat (HF) diet. Wistar rats were fed a HF diet for 6 weeks. During the last 3 weeks of the experiment, the animals fed the HF diet were divided into two groups, either

    Journal: British Journal of Pharmacology

    Article Title: Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

    doi: 10.1111/j.1476-5381.2011.01716.x

    Figure Lengend Snippet: Chronic Sirolimus administration exacerbates glucose intolerance of rats fed a high-fat (HF) diet. Wistar rats were fed a HF diet for 6 weeks. During the last 3 weeks of the experiment, the animals fed the HF diet were divided into two groups, either

    Article Snippet: Sirolimus is a clinically formulated injectable form of rapamycin, which contains, in addition to rapamycin, other inactive components (0.1% sodium CMC, 0.25% Polysobate 80) and was kindly provided by Wyeth Pharma GmbH (Munster, Germany).

    Techniques:

    Chronic mTOR inhibition by rapamycin impairs glucose uptake, glycogen synthesis, GLUT transporters expression and translocation to the plasma membrane in response to insulin in L6 cells. L6 myotubes were exposed for 48 h to either DMSO (CTL) or 10–100

    Journal: British Journal of Pharmacology

    Article Title: Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

    doi: 10.1111/j.1476-5381.2011.01716.x

    Figure Lengend Snippet: Chronic mTOR inhibition by rapamycin impairs glucose uptake, glycogen synthesis, GLUT transporters expression and translocation to the plasma membrane in response to insulin in L6 cells. L6 myotubes were exposed for 48 h to either DMSO (CTL) or 10–100

    Article Snippet: Sirolimus is a clinically formulated injectable form of rapamycin, which contains, in addition to rapamycin, other inactive components (0.1% sodium CMC, 0.25% Polysobate 80) and was kindly provided by Wyeth Pharma GmbH (Munster, Germany).

    Techniques: Inhibition, Expressing, Translocation Assay, CTL Assay

    Chronic Sirolimus administration decreases body weight gain, food intake and fat mass of Wistar rats fed a standard diet. Wistar rats were chronically administered for 3 weeks with either vehicle or Sirolimus (2 mg·kg −1 ·day −1

    Journal: British Journal of Pharmacology

    Article Title: Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

    doi: 10.1111/j.1476-5381.2011.01716.x

    Figure Lengend Snippet: Chronic Sirolimus administration decreases body weight gain, food intake and fat mass of Wistar rats fed a standard diet. Wistar rats were chronically administered for 3 weeks with either vehicle or Sirolimus (2 mg·kg −1 ·day −1

    Article Snippet: Sirolimus is a clinically formulated injectable form of rapamycin, which contains, in addition to rapamycin, other inactive components (0.1% sodium CMC, 0.25% Polysobate 80) and was kindly provided by Wyeth Pharma GmbH (Munster, Germany).

    Techniques:

    Chronic mTOR inhibition by rapamycin alters Akt activation by insulin in L6 myotubes. L6 myotubes were exposed for 48 h to either DMSO (CTL) or 10–100 nM rapamycin (RAPA) before analyses. (A) Representative Western blots of total and phosphorylated

    Journal: British Journal of Pharmacology

    Article Title: Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

    doi: 10.1111/j.1476-5381.2011.01716.x

    Figure Lengend Snippet: Chronic mTOR inhibition by rapamycin alters Akt activation by insulin in L6 myotubes. L6 myotubes were exposed for 48 h to either DMSO (CTL) or 10–100 nM rapamycin (RAPA) before analyses. (A) Representative Western blots of total and phosphorylated

    Article Snippet: Sirolimus is a clinically formulated injectable form of rapamycin, which contains, in addition to rapamycin, other inactive components (0.1% sodium CMC, 0.25% Polysobate 80) and was kindly provided by Wyeth Pharma GmbH (Munster, Germany).

    Techniques: Inhibition, Activation Assay, CTL Assay, Western Blot

    Chronic mTOR inhibition induces glucose intolerance and muscle insulin resistance in Wistar rats fed a standard diet. Wistar rats were chronically administered with either vehicle or Sirolimus (2 mg·kg −1 ·day −1 ). (A) Glycaemia

    Journal: British Journal of Pharmacology

    Article Title: Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

    doi: 10.1111/j.1476-5381.2011.01716.x

    Figure Lengend Snippet: Chronic mTOR inhibition induces glucose intolerance and muscle insulin resistance in Wistar rats fed a standard diet. Wistar rats were chronically administered with either vehicle or Sirolimus (2 mg·kg −1 ·day −1 ). (A) Glycaemia

    Article Snippet: Sirolimus is a clinically formulated injectable form of rapamycin, which contains, in addition to rapamycin, other inactive components (0.1% sodium CMC, 0.25% Polysobate 80) and was kindly provided by Wyeth Pharma GmbH (Munster, Germany).

    Techniques: Inhibition

    Chronic mTOR inhibition by Sirolimus induces hyperglycaemia, glucose intolerance and insulin resistance

    Journal: British Journal of Pharmacology

    Article Title: Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats

    doi: 10.1111/j.1476-5381.2011.01716.x

    Figure Lengend Snippet: Chronic mTOR inhibition by Sirolimus induces hyperglycaemia, glucose intolerance and insulin resistance

    Article Snippet: Sirolimus is a clinically formulated injectable form of rapamycin, which contains, in addition to rapamycin, other inactive components (0.1% sodium CMC, 0.25% Polysobate 80) and was kindly provided by Wyeth Pharma GmbH (Munster, Germany).

    Techniques: Inhibition

    PINK1 regulation by chloroquine and rapamycin. Identification of PINK1 ( a ) whole lysate from rat brain cells was used as positive control for protein identification. 15 μg of proteins from rat brain and human spermatozoa were loaded, separated by SDS-PAGE and immunoblotting was performed with a specific antibody against PINK1. PINK1, green signal, was studied by immunofluorescence, as described in material and methods in fresh samples ( b ). Regulation of PINK1 protein was studied in fresh and after 2 hours of incubation ( c ) and in the presence of chloroquine (50 μM) and rapamycin (100 nM) ( d ). Proteins were extracted, resolved and detected with specific antibody. Data from PINK1 was normalized respect to α-tubulin. Columns with asterisk indicate significant differences (P

    Journal: Scientific Reports

    Article Title: Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility

    doi: 10.1038/srep33647

    Figure Lengend Snippet: PINK1 regulation by chloroquine and rapamycin. Identification of PINK1 ( a ) whole lysate from rat brain cells was used as positive control for protein identification. 15 μg of proteins from rat brain and human spermatozoa were loaded, separated by SDS-PAGE and immunoblotting was performed with a specific antibody against PINK1. PINK1, green signal, was studied by immunofluorescence, as described in material and methods in fresh samples ( b ). Regulation of PINK1 protein was studied in fresh and after 2 hours of incubation ( c ) and in the presence of chloroquine (50 μM) and rapamycin (100 nM) ( d ). Proteins were extracted, resolved and detected with specific antibody. Data from PINK1 was normalized respect to α-tubulin. Columns with asterisk indicate significant differences (P

    Article Snippet: Rapamycin (#553211) from MERCK (Billerica, MA).

    Techniques: Positive Control, SDS Page, Immunofluorescence, Incubation

    Caspase 3/7 activation. Sperm cells were incubated in presence of chloroquine (50 μM) and rapamycin (500 nM) for 2 hours at 37 °C. After the incubation sperm cells were incubated with CellEvent Caspase-3/7 Green Detection Reagent, ethidium homodimer (Eth) and Hoechst 33342 and examined by flow cytometry as described in material and methods. Graphic shows the percentage of Caspase 3/7 positive cells and Eth negative cells (dead cells), expressed as increase of active caspase 3/7 in treatments respect to control (containing vehicle). Columns with asterisk indicate significant differences (P

    Journal: Scientific Reports

    Article Title: Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility

    doi: 10.1038/srep33647

    Figure Lengend Snippet: Caspase 3/7 activation. Sperm cells were incubated in presence of chloroquine (50 μM) and rapamycin (500 nM) for 2 hours at 37 °C. After the incubation sperm cells were incubated with CellEvent Caspase-3/7 Green Detection Reagent, ethidium homodimer (Eth) and Hoechst 33342 and examined by flow cytometry as described in material and methods. Graphic shows the percentage of Caspase 3/7 positive cells and Eth negative cells (dead cells), expressed as increase of active caspase 3/7 in treatments respect to control (containing vehicle). Columns with asterisk indicate significant differences (P

    Article Snippet: Rapamycin (#553211) from MERCK (Billerica, MA).

    Techniques: Activation Assay, Incubation, Flow Cytometry, Cytometry

    Intracellular pH, ATP and calcium concentration. Intracellular pH ( a ), ATP concentration ( b ) and intracellular calcium concentration ( c1,c2 ) were studied in human spermatozoa, as described in material and methods section, after 2 hours of incubation with chloroquine (50 μM) and rapamycin (100 nM). ATP results are expressed as pM concentration of ATP in 100 μg of protein in each sample. Fluorescence of FURA-2-AM was recorded and changes in intracellular [Ca 2+ ]i were monitored every second ( c1 ). Area under the curve after progesterone addition was calculated and expressed as percentage of treatment vs control ( c2 ). Results are expressed as mean ± SEM from 4 independent experiments. Columns marked with *indicate significant differences compared to control (containing vehicle) (P

    Journal: Scientific Reports

    Article Title: Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility

    doi: 10.1038/srep33647

    Figure Lengend Snippet: Intracellular pH, ATP and calcium concentration. Intracellular pH ( a ), ATP concentration ( b ) and intracellular calcium concentration ( c1,c2 ) were studied in human spermatozoa, as described in material and methods section, after 2 hours of incubation with chloroquine (50 μM) and rapamycin (100 nM). ATP results are expressed as pM concentration of ATP in 100 μg of protein in each sample. Fluorescence of FURA-2-AM was recorded and changes in intracellular [Ca 2+ ]i were monitored every second ( c1 ). Area under the curve after progesterone addition was calculated and expressed as percentage of treatment vs control ( c2 ). Results are expressed as mean ± SEM from 4 independent experiments. Columns marked with *indicate significant differences compared to control (containing vehicle) (P

    Article Snippet: Rapamycin (#553211) from MERCK (Billerica, MA).

    Techniques: Concentration Assay, Incubation, Fluorescence

    Effect of chloroquine and rapamycin on LC3-I and LC3-II expression. Human spermatozoa were incubated for 2 hours at 37 °C in presence or absence of chloroquine ( a ) and rapamycin ( b ). Proteins were extracted and resolved by SDS_PAGE. Immunoblotting was performed with anti-LC3 antibody (described in materials and methods section). Results are expressed as increase of LC3-II/LC3-I ratio respect to control samples (containing only vehicle). Columns with asterisk indicate significant differences (P

    Journal: Scientific Reports

    Article Title: Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility

    doi: 10.1038/srep33647

    Figure Lengend Snippet: Effect of chloroquine and rapamycin on LC3-I and LC3-II expression. Human spermatozoa were incubated for 2 hours at 37 °C in presence or absence of chloroquine ( a ) and rapamycin ( b ). Proteins were extracted and resolved by SDS_PAGE. Immunoblotting was performed with anti-LC3 antibody (described in materials and methods section). Results are expressed as increase of LC3-II/LC3-I ratio respect to control samples (containing only vehicle). Columns with asterisk indicate significant differences (P

    Article Snippet: Rapamycin (#553211) from MERCK (Billerica, MA).

    Techniques: Expressing, Incubation, SDS Page

    TOM20 protein and MitoTacker Deep Red (MTDR) fluorescence regulated by chloroquine and rapamycin. Regulation of TOM20 protein was studied in fresh and after 2 hours of incubation ( a ) and in the presence of chloroquine (50 μM) and rapamycin (100 nM) ( b ). Proteins were extracted, resolved and detected with specific antibody. Data from TOM20 was normalized respect to α-tubulin. Columns with asterisk indicate significant differences (P

    Journal: Scientific Reports

    Article Title: Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility

    doi: 10.1038/srep33647

    Figure Lengend Snippet: TOM20 protein and MitoTacker Deep Red (MTDR) fluorescence regulated by chloroquine and rapamycin. Regulation of TOM20 protein was studied in fresh and after 2 hours of incubation ( a ) and in the presence of chloroquine (50 μM) and rapamycin (100 nM) ( b ). Proteins were extracted, resolved and detected with specific antibody. Data from TOM20 was normalized respect to α-tubulin. Columns with asterisk indicate significant differences (P

    Article Snippet: Rapamycin (#553211) from MERCK (Billerica, MA).

    Techniques: Fluorescence, Incubation

    Regulation of AMPKα 1/2 phosphorylation by chloroquine and rapamycin. Spermatozoa were incubated in presence of chloroquine (50 μM) and rapamycin (100 nM). Proteins were then extracted and analyzed by immunoblotting. AMPKα 1/2 phosphorylation was studied with a phosphospecific antibody that recognized phosphorylation on Thr 172. These membranes were also incubated with tubulin for normalization. Results represent the fold-increase of P-Thr 172 -AMPKα 1/2 normalized with tubulin. Columns with asterisk indicate significant differences (P

    Journal: Scientific Reports

    Article Title: Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility

    doi: 10.1038/srep33647

    Figure Lengend Snippet: Regulation of AMPKα 1/2 phosphorylation by chloroquine and rapamycin. Spermatozoa were incubated in presence of chloroquine (50 μM) and rapamycin (100 nM). Proteins were then extracted and analyzed by immunoblotting. AMPKα 1/2 phosphorylation was studied with a phosphospecific antibody that recognized phosphorylation on Thr 172. These membranes were also incubated with tubulin for normalization. Results represent the fold-increase of P-Thr 172 -AMPKα 1/2 normalized with tubulin. Columns with asterisk indicate significant differences (P

    Article Snippet: Rapamycin (#553211) from MERCK (Billerica, MA).

    Techniques: Incubation

    Effect of chloroquine and rapamycin on spermatozoa viability and motility. Sperm cells were incubated in presence of chloroquine (50 μM) and rapamycin (500 nM) for 2 hours at 37 °C. Further, cells were incubated with SYBR 14, propidium iodide (PI) and Hoechst 33342 and examined by flow cytometry (described in material and methods). Sperm motility was assessed by CASA. ( a ) graphic shows the percentage of SYBR 14 positive and PI negative cells and results are expressed as the increase respect to control ± SEM (containing vehicle) (n = 6); ( b ) figures represent the percentage of spermatozoa with progressive and rapid motility. Results are represented as percentage of maximum ± SEM (n = 4). Columns with asterisk indicate significant differences (P

    Journal: Scientific Reports

    Article Title: Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility

    doi: 10.1038/srep33647

    Figure Lengend Snippet: Effect of chloroquine and rapamycin on spermatozoa viability and motility. Sperm cells were incubated in presence of chloroquine (50 μM) and rapamycin (500 nM) for 2 hours at 37 °C. Further, cells were incubated with SYBR 14, propidium iodide (PI) and Hoechst 33342 and examined by flow cytometry (described in material and methods). Sperm motility was assessed by CASA. ( a ) graphic shows the percentage of SYBR 14 positive and PI negative cells and results are expressed as the increase respect to control ± SEM (containing vehicle) (n = 6); ( b ) figures represent the percentage of spermatozoa with progressive and rapid motility. Results are represented as percentage of maximum ± SEM (n = 4). Columns with asterisk indicate significant differences (P

    Article Snippet: Rapamycin (#553211) from MERCK (Billerica, MA).

    Techniques: Incubation, Flow Cytometry, Cytometry

    The RLR-mediated production of IFN-α and pro-inflammatory cytokines is decreased upon mTOR blockade in moDCs. Immature moDCs were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h and then stimulated with 3p-hpRNA (0.5 μg/ml) (A) , polyI:C (1 μg/ml) (B) or VSV (MOI 1) (C) . (A–C) IFN-α, IL-6 and TNF protein levels were measured by ELISA 24 h after 3p-hpRNA and polyI:C activation as well as 18 h after VSV stimulation. Data are shown as mean ± SD from 5 to 8 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: The RLR-mediated production of IFN-α and pro-inflammatory cytokines is decreased upon mTOR blockade in moDCs. Immature moDCs were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h and then stimulated with 3p-hpRNA (0.5 μg/ml) (A) , polyI:C (1 μg/ml) (B) or VSV (MOI 1) (C) . (A–C) IFN-α, IL-6 and TNF protein levels were measured by ELISA 24 h after 3p-hpRNA and polyI:C activation as well as 18 h after VSV stimulation. Data are shown as mean ± SD from 5 to 8 independent experiments. * p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay

    The RLR-stimulated secretion of IFN-α and pro-inflammatory cytokines is abolished upon mTOR blockade in primary human pDCs. Primary pDCs were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h and then stimulated with 3p-hpRNA (0.5 μg/ml) (A) or polyI:C (1 μg/ml) (B) . (A,B) IFN-α, IL-6 and TNF protein levels were measured by ELISA 6 h after stimulation. Data are shown as mean ± SD from 3 independent experiments. ** p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: The RLR-stimulated secretion of IFN-α and pro-inflammatory cytokines is abolished upon mTOR blockade in primary human pDCs. Primary pDCs were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h and then stimulated with 3p-hpRNA (0.5 μg/ml) (A) or polyI:C (1 μg/ml) (B) . (A,B) IFN-α, IL-6 and TNF protein levels were measured by ELISA 6 h after stimulation. Data are shown as mean ± SD from 3 independent experiments. ** p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Enzyme-linked Immunosorbent Assay

    The T cell activating capacity of RLR-stimulated primary human pDCs is decreased by rapamycin but not AZD8055 pre-treatment. Naïve CD8 + T cells were co-cultured with allogeneic primary human pDCs pre-treated with the indicated reagents. After 6 days of co-cultivation T cells were stimulated with phorbol myristate acetate (0.1 μg/ml) and ionomycin (1 μg/ml) in the presence of monensin for 5 h. IFN-y and Granzyme B production of CD8 + T cells was measured by intracellular cytokine staining and flow cytometry. (A,C,E,G) Data from one representative experiment are shown. Numbers in each quadrant represent the percentage of cells. In A and E as well as in C and G T cell controls are the same, since representative dot plots originate from the same set of experiment from one single donor. (B,D,F,H) Bar graphs represent the mean ± SD of 6 independent experiments. *** p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: The T cell activating capacity of RLR-stimulated primary human pDCs is decreased by rapamycin but not AZD8055 pre-treatment. Naïve CD8 + T cells were co-cultured with allogeneic primary human pDCs pre-treated with the indicated reagents. After 6 days of co-cultivation T cells were stimulated with phorbol myristate acetate (0.1 μg/ml) and ionomycin (1 μg/ml) in the presence of monensin for 5 h. IFN-y and Granzyme B production of CD8 + T cells was measured by intracellular cytokine staining and flow cytometry. (A,C,E,G) Data from one representative experiment are shown. Numbers in each quadrant represent the percentage of cells. In A and E as well as in C and G T cell controls are the same, since representative dot plots originate from the same set of experiment from one single donor. (B,D,F,H) Bar graphs represent the mean ± SD of 6 independent experiments. *** p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Cell Culture, Staining, Flow Cytometry

    Rapamycin but not AZD8055 effectively inhibits the ability of RLR-stimulated moDCs and primary pDCs to induce proliferation of naive T cells. CFSE-labeled naïve CD8 + T cells were co-cultured with allogeneic moDCs (A–D) and primary pDCs (E–H) pre-treated with the indicated reagents. After 5 days of co-cultivation, cell division was measured by flow cytometry. (A,C,E,G) Representative histograms are shown where numbers indicate the percentage of viable dividing T cells. In A and C T cell controls are the same, since representative dot plots originate from the same set of experiment from one single donor. (B,D,F,H) Bar graphs represent the mean ± SD of at least 6 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: Rapamycin but not AZD8055 effectively inhibits the ability of RLR-stimulated moDCs and primary pDCs to induce proliferation of naive T cells. CFSE-labeled naïve CD8 + T cells were co-cultured with allogeneic moDCs (A–D) and primary pDCs (E–H) pre-treated with the indicated reagents. After 5 days of co-cultivation, cell division was measured by flow cytometry. (A,C,E,G) Representative histograms are shown where numbers indicate the percentage of viable dividing T cells. In A and C T cell controls are the same, since representative dot plots originate from the same set of experiment from one single donor. (B,D,F,H) Bar graphs represent the mean ± SD of at least 6 independent experiments. * p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Labeling, Cell Culture, Flow Cytometry

    The RLR-mediated shift to glycolysis is impaired upon mTOR inhibition in moDCs. Immature moDCs were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h and then stimulated with 3p-hpRNA (0.5 μg/ml) (A,C) or polyI:C (1 μg/ml) (B,D) for 12 h. (A,B) The expression of LDHA , HK2 and HIF1A was assessed at the mRNA level by real-time PCR. Bar graphs represent the mean ± SD of 4 independent experiments. (C,D) Lactate concentrations were measured from cell culture supernatants. Bar graphs represent the mean ± SD of 6 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: The RLR-mediated shift to glycolysis is impaired upon mTOR inhibition in moDCs. Immature moDCs were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h and then stimulated with 3p-hpRNA (0.5 μg/ml) (A,C) or polyI:C (1 μg/ml) (B,D) for 12 h. (A,B) The expression of LDHA , HK2 and HIF1A was assessed at the mRNA level by real-time PCR. Bar graphs represent the mean ± SD of 4 independent experiments. (C,D) Lactate concentrations were measured from cell culture supernatants. Bar graphs represent the mean ± SD of 6 independent experiments. * p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Cell Culture

    RLR stimulation increases mTORC1 and mTORC2 activity in GEN2.2 cells that can be effectively inhibited by rapamycin and AZD8055 pre-conditioning. GEN2.2 cells were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h and then stimulated with 3p-hpRNA (0.5 μg/ml) (A,B) or polyI:C (1 μg/ml) (C,D) in a time-dependent manner. Kinetics of p70S6K and Akt phosphorylation was determined by western blotting. (A,C) Representative blots are shown. (B,D) Bar graphs represent the mean ± SD from 4 independent experiments. Specific bands for Akt are indicated by arrows. * p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: RLR stimulation increases mTORC1 and mTORC2 activity in GEN2.2 cells that can be effectively inhibited by rapamycin and AZD8055 pre-conditioning. GEN2.2 cells were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h and then stimulated with 3p-hpRNA (0.5 μg/ml) (A,B) or polyI:C (1 μg/ml) (C,D) in a time-dependent manner. Kinetics of p70S6K and Akt phosphorylation was determined by western blotting. (A,C) Representative blots are shown. (B,D) Bar graphs represent the mean ± SD from 4 independent experiments. Specific bands for Akt are indicated by arrows. * p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Activity Assay, Western Blot

    RLR stimulation enhances TBK1 activity in moDCs that is decreased by rapamycin and AZD8055 pre-treatment. Immature moDCs were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h then stimulated with 3p-hpRNA (0.5 μg/ml) (A,C) or polyI:C (1 μg/ml) (B,D) for different time periods. Kinetics of TBK1 phosphorylation were determined by western blotting. (A,B) Representative blots are shown. (C,D) Bar graphs represent the mean ± SD of at least 3 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: RLR stimulation enhances TBK1 activity in moDCs that is decreased by rapamycin and AZD8055 pre-treatment. Immature moDCs were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h then stimulated with 3p-hpRNA (0.5 μg/ml) (A,C) or polyI:C (1 μg/ml) (B,D) for different time periods. Kinetics of TBK1 phosphorylation were determined by western blotting. (A,B) Representative blots are shown. (C,D) Bar graphs represent the mean ± SD of at least 3 independent experiments. * p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Activity Assay, Western Blot

    The RLR-mediated production of IFN-α and pro-inflammatory cytokines is decreased upon mTOR inhibition in GEN2.2 cells. GEN2.2 cells were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h then stimulated with 3p-hpRNA (0.5 μg/ml) (A) , polyI:C (1 μg/ml) (B) , or VSV (MOI 1) (C) . (A–C) IFN-α, IL-6 and TNF protein levels were measured by ELISA 6 h after 3p-hpRNA as well as polyI:C activation and 18 h after VSV stimulation. Data are shown as mean ± SD from 4 to 8 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: The RLR-mediated production of IFN-α and pro-inflammatory cytokines is decreased upon mTOR inhibition in GEN2.2 cells. GEN2.2 cells were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h then stimulated with 3p-hpRNA (0.5 μg/ml) (A) , polyI:C (1 μg/ml) (B) , or VSV (MOI 1) (C) . (A–C) IFN-α, IL-6 and TNF protein levels were measured by ELISA 6 h after 3p-hpRNA as well as polyI:C activation and 18 h after VSV stimulation. Data are shown as mean ± SD from 4 to 8 independent experiments. * p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Activation Assay

    RLR stimulation increases mTORC1 and mTORC2 activity in moDCs that is effectively inhibited by rapamycin and AZD8055 pre-treatment. Immature moDCs were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h then stimulated with 3p-hpRNA (0.5 μg/ml) (A,B) or polyI:C (1 μg/ml) (C,D) for different time periods. Kinetics of p70S6K and Akt phosphorylation were determined by western blotting. (A,C) Representative blots are shown. (B,D) Bar graphs represent the mean ± SD of at least 3 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: RLR stimulation increases mTORC1 and mTORC2 activity in moDCs that is effectively inhibited by rapamycin and AZD8055 pre-treatment. Immature moDCs were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h then stimulated with 3p-hpRNA (0.5 μg/ml) (A,B) or polyI:C (1 μg/ml) (C,D) for different time periods. Kinetics of p70S6K and Akt phosphorylation were determined by western blotting. (A,C) Representative blots are shown. (B,D) Bar graphs represent the mean ± SD of at least 3 independent experiments. * p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Activity Assay, Western Blot

    Rapamycin but not AZD8055 decreased the T cell activating capacity of RLR-activated moDCs. Naïve CD8 + T cells were co-cultured with allogeneic moDCs pre-treated with the indicated reagents. After 6 days of co-cultivation T cells were stimulated with phorbol myristate acetate (0.1 μg/ml) and ionomycin (1 μg/ml) in the presence of monensin for 5 h. IFN-y and Granzyme B production of CD8 + T cells was measured by intracellular cytokine staining using flow cytometry. (A,C,E,G) Data from one representative experiment are shown. Numbers in quadrants indicate percent cells in each. In A and E as well as in C and G T cell controls are the same, since representative dot plots originate from the same set of experiment from one single donor. (B,D,F,H) Bar graphs represent the mean ± SD of 6 independent experiments. *** p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: Rapamycin but not AZD8055 decreased the T cell activating capacity of RLR-activated moDCs. Naïve CD8 + T cells were co-cultured with allogeneic moDCs pre-treated with the indicated reagents. After 6 days of co-cultivation T cells were stimulated with phorbol myristate acetate (0.1 μg/ml) and ionomycin (1 μg/ml) in the presence of monensin for 5 h. IFN-y and Granzyme B production of CD8 + T cells was measured by intracellular cytokine staining using flow cytometry. (A,C,E,G) Data from one representative experiment are shown. Numbers in quadrants indicate percent cells in each. In A and E as well as in C and G T cell controls are the same, since representative dot plots originate from the same set of experiment from one single donor. (B,D,F,H) Bar graphs represent the mean ± SD of 6 independent experiments. *** p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Cell Culture, Staining, Flow Cytometry

    RLR stimulation increases the phosphorylation of TBK1 that is reduced by rapamycin and AZD8055 pre-treatment in GEN2.2 cells. Cells were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h then stimulated with 3p-hpRNA (0.5 μg/ml) (A,C) or polyI:C (1 μg/ml) (B,D) for different time periods. Kinetics of TBK1 phosphorylation were determined by western blotting. (A,B) Representative blots are shown. (C,D) Bar graphs represent the mean ± SD of at least 3 independent experiments. ** p

    Journal: Frontiers in Immunology

    Article Title: Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR

    doi: 10.3389/fimmu.2020.572960

    Figure Lengend Snippet: RLR stimulation increases the phosphorylation of TBK1 that is reduced by rapamycin and AZD8055 pre-treatment in GEN2.2 cells. Cells were pre-treated with vehicle control, 100 nM rapamycin (RAPA) or 100 nM AZD8055 (AZD) for 2 h then stimulated with 3p-hpRNA (0.5 μg/ml) (A,C) or polyI:C (1 μg/ml) (B,D) for different time periods. Kinetics of TBK1 phosphorylation were determined by western blotting. (A,B) Representative blots are shown. (C,D) Bar graphs represent the mean ± SD of at least 3 independent experiments. ** p

    Article Snippet: Cell Stimulation Before stimulation of moDCs half of the medium was removed and replaced by fresh complete medium supplemented with rapamycin (Merck Millipore, Darmstadt, Germany) or AZD8055 (Selleckchem, Houston, TX, United States) at a final concentration of 100 nM.

    Techniques: Western Blot

    Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were transfected with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were transfected with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0

    Article Snippet: qPCR array of autophagy gene expression The effect of silencing PRKCE gene and rapamycin (300 nM) and 3-MA (5 mM) treatment for 24 h on the expression of genes involved in the autophagy pathways was assessed using Real Time Primers ready Human Autophagy Primer Library 96 (HATPL-1) (Biomol GmbH, Hamburg, Germany).

    Techniques: Immunofluorescence, Transfection, Imaging

    Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Article Snippet: qPCR array of autophagy gene expression The effect of silencing PRKCE gene and rapamycin (300 nM) and 3-MA (5 mM) treatment for 24 h on the expression of genes involved in the autophagy pathways was assessed using Real Time Primers ready Human Autophagy Primer Library 96 (HATPL-1) (Biomol GmbH, Hamburg, Germany).

    Techniques: Transfection

    Changes in autophagy gene expression in U-138 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-138 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Changes in autophagy gene expression in U-138 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-138 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Article Snippet: qPCR array of autophagy gene expression The effect of silencing PRKCE gene and rapamycin (300 nM) and 3-MA (5 mM) treatment for 24 h on the expression of genes involved in the autophagy pathways was assessed using Real Time Primers ready Human Autophagy Primer Library 96 (HATPL-1) (Biomol GmbH, Hamburg, Germany).

    Techniques: Expressing, Transfection

    Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: qPCR array of autophagy gene expression The effect of silencing PRKCE gene and rapamycin (300 nM) and 3-MA (5 mM) treatment for 24 h on the expression of genes involved in the autophagy pathways was assessed using Real Time Primers ready Human Autophagy Primer Library 96 (HATPL-1) (Biomol GmbH, Hamburg, Germany).

    Techniques: Activation Assay, Transfection, Construct

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: qPCR array of autophagy gene expression The effect of silencing PRKCE gene and rapamycin (300 nM) and 3-MA (5 mM) treatment for 24 h on the expression of genes involved in the autophagy pathways was assessed using Real Time Primers ready Human Autophagy Primer Library 96 (HATPL-1) (Biomol GmbH, Hamburg, Germany).

    Techniques: Transfection

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: qPCR array of autophagy gene expression The effect of silencing PRKCE gene and rapamycin (300 nM) and 3-MA (5 mM) treatment for 24 h on the expression of genes involved in the autophagy pathways was assessed using Real Time Primers ready Human Autophagy Primer Library 96 (HATPL-1) (Biomol GmbH, Hamburg, Germany).

    Techniques: Transfection

    Changes in autophagy gene expression in U-118 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-118 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Changes in autophagy gene expression in U-118 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-118 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Article Snippet: qPCR array of autophagy gene expression The effect of silencing PRKCE gene and rapamycin (300 nM) and 3-MA (5 mM) treatment for 24 h on the expression of genes involved in the autophagy pathways was assessed using Real Time Primers ready Human Autophagy Primer Library 96 (HATPL-1) (Biomol GmbH, Hamburg, Germany).

    Techniques: Expressing, Transfection

    Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: qPCR array of autophagy gene expression The effect of silencing PRKCE gene and rapamycin (300 nM) and 3-MA (5 mM) treatment for 24 h on the expression of genes involved in the autophagy pathways was assessed using Real Time Primers ready Human Autophagy Primer Library 96 (HATPL-1) (Biomol GmbH, Hamburg, Germany).

    Techniques: Transfection

    mTORC1 downregulates PDGFRɑ/AKT pathway through inhibition of FOXO3a ( A ) Tsc2−/− or Tsc1−/− MEFs were infected with lentivirus harboring a vector encoding FOXO3aTM (LV-FOXO3aTM) or the empty vector (LV). ( B ) Tsc2+/+ or Tsc1+/+ MEFs were transfected with two independent siRNAs targeting FOXO3a or the control siRNAs (siNC) for 48 h. A and B. Cell lysates and RNA were subjected to immunoblotting (upper panels) and qRT-PCR (lower panels), respectively. ( C ) Schematic representation of the putative wild-type (WT) and mutated (mut 1 and mut 2 ) FOXO3a-binding sites in the promoter of mouse PDGFRɑ gene. ( D ) HEK293T cells were co-transfected with pPDGFRɑ-Luc reporter plasmid plus FOXO3aTM expression vector (pLVX-FOXO3aTM) or control vector (pLVX) and the internal control plasmid pRL-TK. ( E ) Tsc2−/− MEFs were co-transfected with pLVX-FOXO3aTM plus pPDGFRɑ-Luc, pPDGFRɑ mut1 -Luc, or pPDGFRɑ mut2 -Luc reporter plasmid and pRL-TK plasmid. D and E. Relative luciferase activity was examined 24 h after transfection. ( F ) Tsc2−/− MEFs were transduced with LV-FOXO3aTM or LV lentiviruses. ( G ) Tsc1+/+, Tsc1−/−, or rapamycin-treated (20 nM 24 h) Tsc1−/− MEFs. F and G. Cells were subjected to ChIP assay using an anti-FOXO3a antibody. Normal rabbit IgG antibody served as the negative control. PCR was performed to amplify regions surrounding the putative FOXO3a-binding regions (PBR1 and PBR2) and a nonspecific FOXO3a-binding region (NBR). Representative data from two independent experiments were shown. ** P

    Journal: Oncotarget

    Article Title: Hyperactivated mTORC1 downregulation of FOXO3a/PDGFRα/AKT cascade restrains tuberous sclerosis complex-associated tumor development

    doi: 10.18632/oncotarget.18963

    Figure Lengend Snippet: mTORC1 downregulates PDGFRɑ/AKT pathway through inhibition of FOXO3a ( A ) Tsc2−/− or Tsc1−/− MEFs were infected with lentivirus harboring a vector encoding FOXO3aTM (LV-FOXO3aTM) or the empty vector (LV). ( B ) Tsc2+/+ or Tsc1+/+ MEFs were transfected with two independent siRNAs targeting FOXO3a or the control siRNAs (siNC) for 48 h. A and B. Cell lysates and RNA were subjected to immunoblotting (upper panels) and qRT-PCR (lower panels), respectively. ( C ) Schematic representation of the putative wild-type (WT) and mutated (mut 1 and mut 2 ) FOXO3a-binding sites in the promoter of mouse PDGFRɑ gene. ( D ) HEK293T cells were co-transfected with pPDGFRɑ-Luc reporter plasmid plus FOXO3aTM expression vector (pLVX-FOXO3aTM) or control vector (pLVX) and the internal control plasmid pRL-TK. ( E ) Tsc2−/− MEFs were co-transfected with pLVX-FOXO3aTM plus pPDGFRɑ-Luc, pPDGFRɑ mut1 -Luc, or pPDGFRɑ mut2 -Luc reporter plasmid and pRL-TK plasmid. D and E. Relative luciferase activity was examined 24 h after transfection. ( F ) Tsc2−/− MEFs were transduced with LV-FOXO3aTM or LV lentiviruses. ( G ) Tsc1+/+, Tsc1−/−, or rapamycin-treated (20 nM 24 h) Tsc1−/− MEFs. F and G. Cells were subjected to ChIP assay using an anti-FOXO3a antibody. Normal rabbit IgG antibody served as the negative control. PCR was performed to amplify regions surrounding the putative FOXO3a-binding regions (PBR1 and PBR2) and a nonspecific FOXO3a-binding region (NBR). Representative data from two independent experiments were shown. ** P

    Article Snippet: For the cell viability assays, Tsc1- or Tsc2-null MEFs were seeded in 96-well plates at a density of 3, 000 cells/well and treated with DMSO, rapamycin, AG1295, or combination of rapamycin and AG1295 for 48 h. Followed by added 10 μl of Cell Counting Kit-8 reagent (Beyotime, China) to per well and incubated the plates for 1 h, the optical density (OD value) of each well was measured at 450 nm.

    Techniques: Inhibition, Infection, Plasmid Preparation, Transfection, Quantitative RT-PCR, Binding Assay, Expressing, Luciferase, Activity Assay, Transduction, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction

    mTORC1 is a negative regulator of FOXO3a ( A ) Total cell lysates from Tsc2+/+, Tsc2−/−, rapamycin-treated (20 nM 24 h) Tsc2−/−, Tsc1+/+, Tsc1−/− and rapamycin-treated (20 nM 24 h) Tsc1−/− MEFs were subjected to immunoblotting with the indicated antibodies. ( B – D ) Tsc2+/+, Tsc2−/−, and rapamycin-treated (20 nM 24 h) Tsc2−/− MEFs. B. The cytoplasm (Cyt) and nuclear (Nuc) proteins were subjected to immunoblotting with the indicated antibodies. C. The expression of FOXO3a was analyzed by an immunofluorescence assay. Scale bar, 50 μm. D. The cells were co-transfected with pGMFOXO-Luc (200 ng) and the internal control plasmid pRL-TK (20 ng). The relative luciferase activity was measured 24 h after transfection. ( E ) Tsc1−/− MEFs were treated with or without rapamycin (20 nM) for 24 h. The nuclear proteins were subjected to immunoblotting with the indicated antibodies (left panel). The relative luciferase activity was measured as D (right panel). ( F ) Total cell lysates from Tsc2−/− or Tsc1−/− MEFs transduced with shRaptor or shSc lentiviruses were subjected to immunoblotting with the indicated antibodies. G-H. Tsc2−/− MEFs were transduced with shRaptor or shSc lentiviruses. ( G ) The cytoplasm (Cyt) and nuclear (Nuc) proteins were subjected to immunoblotting with the indicated antibodies. ( H ) The cells were co-transfected with pGMFOXO-Luc (200 ng) and the internal control plasmid pRL-TK (20 ng). The relative luciferase activity was measured 24 h after transfection. ( I ) Tsc1−/− MEFs were infected with shRaptor or shSc lentiviruses. The nuclear proteins were subjected to immunoblotting with the indicated antibodies (left panel). The relative luciferase activity was measured as H (right panel). ** P

    Journal: Oncotarget

    Article Title: Hyperactivated mTORC1 downregulation of FOXO3a/PDGFRα/AKT cascade restrains tuberous sclerosis complex-associated tumor development

    doi: 10.18632/oncotarget.18963

    Figure Lengend Snippet: mTORC1 is a negative regulator of FOXO3a ( A ) Total cell lysates from Tsc2+/+, Tsc2−/−, rapamycin-treated (20 nM 24 h) Tsc2−/−, Tsc1+/+, Tsc1−/− and rapamycin-treated (20 nM 24 h) Tsc1−/− MEFs were subjected to immunoblotting with the indicated antibodies. ( B – D ) Tsc2+/+, Tsc2−/−, and rapamycin-treated (20 nM 24 h) Tsc2−/− MEFs. B. The cytoplasm (Cyt) and nuclear (Nuc) proteins were subjected to immunoblotting with the indicated antibodies. C. The expression of FOXO3a was analyzed by an immunofluorescence assay. Scale bar, 50 μm. D. The cells were co-transfected with pGMFOXO-Luc (200 ng) and the internal control plasmid pRL-TK (20 ng). The relative luciferase activity was measured 24 h after transfection. ( E ) Tsc1−/− MEFs were treated with or without rapamycin (20 nM) for 24 h. The nuclear proteins were subjected to immunoblotting with the indicated antibodies (left panel). The relative luciferase activity was measured as D (right panel). ( F ) Total cell lysates from Tsc2−/− or Tsc1−/− MEFs transduced with shRaptor or shSc lentiviruses were subjected to immunoblotting with the indicated antibodies. G-H. Tsc2−/− MEFs were transduced with shRaptor or shSc lentiviruses. ( G ) The cytoplasm (Cyt) and nuclear (Nuc) proteins were subjected to immunoblotting with the indicated antibodies. ( H ) The cells were co-transfected with pGMFOXO-Luc (200 ng) and the internal control plasmid pRL-TK (20 ng). The relative luciferase activity was measured 24 h after transfection. ( I ) Tsc1−/− MEFs were infected with shRaptor or shSc lentiviruses. The nuclear proteins were subjected to immunoblotting with the indicated antibodies (left panel). The relative luciferase activity was measured as H (right panel). ** P

    Article Snippet: For the cell viability assays, Tsc1- or Tsc2-null MEFs were seeded in 96-well plates at a density of 3, 000 cells/well and treated with DMSO, rapamycin, AG1295, or combination of rapamycin and AG1295 for 48 h. Followed by added 10 μl of Cell Counting Kit-8 reagent (Beyotime, China) to per well and incubated the plates for 1 h, the optical density (OD value) of each well was measured at 450 nm.

    Techniques: Expressing, Immunofluorescence, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Transduction, Infection

    The combination of rapamycin and AG1295 more effectively suppresses the growth of cells lacking TSC1/TSC2 complex in vitro and in vivo than either agent alone ( A , B ) Tsc2−/− (A) or Tsc1−/− MEFs (B) were treated with a combination of 5 nM rapamycin (Rapa) and 20 μM AG1295 or either agent alone for 48 h. Cell viability was examined with an MTT assay. ( C – F ) NTC/T2-null cells were inoculated subcutaneously into the nude mice to evaluate the effects of rapamycin and AG1295 in vivo . (C) Tumor volume growth curves. (D) Dissected tumors. (E) Tumor weight. (F) Body weights of mice. ** P

    Journal: Oncotarget

    Article Title: Hyperactivated mTORC1 downregulation of FOXO3a/PDGFRα/AKT cascade restrains tuberous sclerosis complex-associated tumor development

    doi: 10.18632/oncotarget.18963

    Figure Lengend Snippet: The combination of rapamycin and AG1295 more effectively suppresses the growth of cells lacking TSC1/TSC2 complex in vitro and in vivo than either agent alone ( A , B ) Tsc2−/− (A) or Tsc1−/− MEFs (B) were treated with a combination of 5 nM rapamycin (Rapa) and 20 μM AG1295 or either agent alone for 48 h. Cell viability was examined with an MTT assay. ( C – F ) NTC/T2-null cells were inoculated subcutaneously into the nude mice to evaluate the effects of rapamycin and AG1295 in vivo . (C) Tumor volume growth curves. (D) Dissected tumors. (E) Tumor weight. (F) Body weights of mice. ** P

    Article Snippet: For the cell viability assays, Tsc1- or Tsc2-null MEFs were seeded in 96-well plates at a density of 3, 000 cells/well and treated with DMSO, rapamycin, AG1295, or combination of rapamycin and AG1295 for 48 h. Followed by added 10 μl of Cell Counting Kit-8 reagent (Beyotime, China) to per well and incubated the plates for 1 h, the optical density (OD value) of each well was measured at 450 nm.

    Techniques: In Vitro, In Vivo, MTT Assay, Mouse Assay

    mTORC1 is a negative regulator of PDGFRα ( A ) Tsc2+/+ and Tsc2−/− MEFs. ( B ) Tsc1+/+ and Tsc1−/− MEFs. ( C , D ) Tsc2−/− (C) or Tsc1−/− MEFs (D) were treated with DMSO or with 20 nM rapamycin (Rapa) for 24 h. ( E , F ) Tsc2−/− (E) or Tsc1−/− MEFs (F) were transfected with the control siRNAs (siNC) or siRNAs targeting Raptor for 48 h. A–F. Cell lysates were subjected to immunoblotting with the indicated antibodies (left panels). qRT-PCR was performed to detect the mRNA level of PDGFRɑ (right panels). ** P

    Journal: Oncotarget

    Article Title: Hyperactivated mTORC1 downregulation of FOXO3a/PDGFRα/AKT cascade restrains tuberous sclerosis complex-associated tumor development

    doi: 10.18632/oncotarget.18963

    Figure Lengend Snippet: mTORC1 is a negative regulator of PDGFRα ( A ) Tsc2+/+ and Tsc2−/− MEFs. ( B ) Tsc1+/+ and Tsc1−/− MEFs. ( C , D ) Tsc2−/− (C) or Tsc1−/− MEFs (D) were treated with DMSO or with 20 nM rapamycin (Rapa) for 24 h. ( E , F ) Tsc2−/− (E) or Tsc1−/− MEFs (F) were transfected with the control siRNAs (siNC) or siRNAs targeting Raptor for 48 h. A–F. Cell lysates were subjected to immunoblotting with the indicated antibodies (left panels). qRT-PCR was performed to detect the mRNA level of PDGFRɑ (right panels). ** P

    Article Snippet: For the cell viability assays, Tsc1- or Tsc2-null MEFs were seeded in 96-well plates at a density of 3, 000 cells/well and treated with DMSO, rapamycin, AG1295, or combination of rapamycin and AG1295 for 48 h. Followed by added 10 μl of Cell Counting Kit-8 reagent (Beyotime, China) to per well and incubated the plates for 1 h, the optical density (OD value) of each well was measured at 450 nm.

    Techniques: Transfection, Quantitative RT-PCR

    Rapamycin (Rapa) and the combination of cyclosporin A (CsA) and Rapa changes β 1 and β 3 integrin expression on MLR-activated cells. Peripheral blood mononuclear cells were cultured for 6 days in the presence of CsA (200 ng per 1 mL medium), Rapa (20 ng per 1 mL medium), and the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR) and then lysed in RIPA buffer. Protein extracts (15 μ g) were digested with endo- β -N-acetylglucosaminidase H (Endo H) from Streptomyces plicatus , SDS-PAGE-separated on 10% gel under reducing conditions, electroblotted onto a PVDF membrane, and probed with specific primary antibodies: mouse monoclonal anti- β 1 (Chemicon, MAB2251, clone B3B11) and rabbit polyclonal anti- β 3 (Chemicon, AB1932). Antibody-bound integrins were visualized by chemiluminescence. GAPDH was the endogenous control. C: untreated cells.

    Journal: Analytical cellular pathology (Amsterdam)

    Article Title: Immunosuppressive Drugs Affect High-Mannose/Hybrid N-Glycans on Human Allostimulated Leukocytes

    doi: 10.1155/2015/324980

    Figure Lengend Snippet: Rapamycin (Rapa) and the combination of cyclosporin A (CsA) and Rapa changes β 1 and β 3 integrin expression on MLR-activated cells. Peripheral blood mononuclear cells were cultured for 6 days in the presence of CsA (200 ng per 1 mL medium), Rapa (20 ng per 1 mL medium), and the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR) and then lysed in RIPA buffer. Protein extracts (15 μ g) were digested with endo- β -N-acetylglucosaminidase H (Endo H) from Streptomyces plicatus , SDS-PAGE-separated on 10% gel under reducing conditions, electroblotted onto a PVDF membrane, and probed with specific primary antibodies: mouse monoclonal anti- β 1 (Chemicon, MAB2251, clone B3B11) and rabbit polyclonal anti- β 3 (Chemicon, AB1932). Antibody-bound integrins were visualized by chemiluminescence. GAPDH was the endogenous control. C: untreated cells.

    Article Snippet: Rapamycin and cyclosporin A were obtained from Wyeth-Lederle and Novartis Pharma, respectively.

    Techniques: Expressing, Cell Culture, SDS Page

    High-mannose/hybrid-type glycans are present mainly on premature β 1 integrin subunit. Cell lysate (L), glycoproteins precipitated with GNA-agarose (P), and supernatant collected after precipitation (S), containing proteins not recognized by GNA, were resolved on 10% SDS-PAGE gel under reducing conditions, electrotransferred to a PVDF membrane and destined for β 1 integrin subunit immunodetection. GAPDH was the endogenous control. C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.

    Journal: Analytical cellular pathology (Amsterdam)

    Article Title: Immunosuppressive Drugs Affect High-Mannose/Hybrid N-Glycans on Human Allostimulated Leukocytes

    doi: 10.1155/2015/324980

    Figure Lengend Snippet: High-mannose/hybrid-type glycans are present mainly on premature β 1 integrin subunit. Cell lysate (L), glycoproteins precipitated with GNA-agarose (P), and supernatant collected after precipitation (S), containing proteins not recognized by GNA, were resolved on 10% SDS-PAGE gel under reducing conditions, electrotransferred to a PVDF membrane and destined for β 1 integrin subunit immunodetection. GAPDH was the endogenous control. C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.

    Article Snippet: Rapamycin and cyclosporin A were obtained from Wyeth-Lederle and Novartis Pharma, respectively.

    Techniques: SDS Page, Immunodetection

    Immunosuppressive drugs alter the surface expression of high-mannose/hybrid N-glycans. Peripheral blood mononuclear cells were cultured for 6 days in the presence of CsA (200 ng per 1 mL medium), Rapa (20 ng per 1 mL medium), and the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR). Immunosuppressive drug-treated and control cells were stained with biotinylated GNA (1 : 100), followed by incubation with FITC-conjugated ExtrAvidin. M2 region corresponds to GNA-positive leukocytes after allostimulation. Fluorescence was measured using a FACSCalibur flow cytometer (BD Biosciences). C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.

    Journal: Analytical cellular pathology (Amsterdam)

    Article Title: Immunosuppressive Drugs Affect High-Mannose/Hybrid N-Glycans on Human Allostimulated Leukocytes

    doi: 10.1155/2015/324980

    Figure Lengend Snippet: Immunosuppressive drugs alter the surface expression of high-mannose/hybrid N-glycans. Peripheral blood mononuclear cells were cultured for 6 days in the presence of CsA (200 ng per 1 mL medium), Rapa (20 ng per 1 mL medium), and the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR). Immunosuppressive drug-treated and control cells were stained with biotinylated GNA (1 : 100), followed by incubation with FITC-conjugated ExtrAvidin. M2 region corresponds to GNA-positive leukocytes after allostimulation. Fluorescence was measured using a FACSCalibur flow cytometer (BD Biosciences). C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.

    Article Snippet: Rapamycin and cyclosporin A were obtained from Wyeth-Lederle and Novartis Pharma, respectively.

    Techniques: Expressing, Cell Culture, Staining, Incubation, Fluorescence, Flow Cytometry, Cytometry

    Immunosuppressive drugs change the intensity of bands containing GNA-positive proteins. Peripheral blood mononuclear cells were cultured for 6 days in the presence of the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR) and then lysed in RIPA buffer. Whole-cell lysate proteins (500 μ g) were precipitated with 25 μ L agarose-bound Galanthus nivalis lectin (GNA). The captured glycoproteins were recovered by boiling in Laemmli sample buffer with β -ME and resolved by 10% SDS-PAGE under reducing conditions. One-fifth of the GNA precipitate was destined for Western blotting (WB) and after probing with biotinylated-GNA was visualized by AP colorimetric reaction (a). The remaining four-fifths of the precipitate, after resolving on the same gel, were stained with Coomassie Brilliant Blue (CBB) and the bands were excised for MS/MS analysis (b). Molecular weight of proteins was assigned using a PageRuler Prestained Protein Ladder (Thermo Scientific, 26616). LP: lectin precipitation, C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.

    Journal: Analytical cellular pathology (Amsterdam)

    Article Title: Immunosuppressive Drugs Affect High-Mannose/Hybrid N-Glycans on Human Allostimulated Leukocytes

    doi: 10.1155/2015/324980

    Figure Lengend Snippet: Immunosuppressive drugs change the intensity of bands containing GNA-positive proteins. Peripheral blood mononuclear cells were cultured for 6 days in the presence of the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR) and then lysed in RIPA buffer. Whole-cell lysate proteins (500 μ g) were precipitated with 25 μ L agarose-bound Galanthus nivalis lectin (GNA). The captured glycoproteins were recovered by boiling in Laemmli sample buffer with β -ME and resolved by 10% SDS-PAGE under reducing conditions. One-fifth of the GNA precipitate was destined for Western blotting (WB) and after probing with biotinylated-GNA was visualized by AP colorimetric reaction (a). The remaining four-fifths of the precipitate, after resolving on the same gel, were stained with Coomassie Brilliant Blue (CBB) and the bands were excised for MS/MS analysis (b). Molecular weight of proteins was assigned using a PageRuler Prestained Protein Ladder (Thermo Scientific, 26616). LP: lectin precipitation, C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.

    Article Snippet: Rapamycin and cyclosporin A were obtained from Wyeth-Lederle and Novartis Pharma, respectively.

    Techniques: Cell Culture, SDS Page, Western Blot, Staining, Mass Spectrometry, Molecular Weight