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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    98
    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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    93
    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: 93/100, 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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    99
    Tocris 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 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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    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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    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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    93
    Cayman Chemical rapamycin
    A77 1726 induces SOD1 G93A co-localization with autophagosomes. RFP-LC3 stably transfected NSC34 cells were transiently transfected with SOD1-GFP ( a ) or SOD1 G93A -GFP ( b ) expression vectors. After incubation for 40 h, the cells were treated with DMSO (0.2%), A77 1726 (200 μM) or <t>rapamycin</t> (50 nM) ( a ) for 24 h. The cells were fixed and examined under a confocal microscope for the localization of autophagosomes (RFP-LC3) and for SOD1-GFP or SOD1 G93A -GFP protein aggregates. c–e NSC34 cells were transfected with control or ATG7 siRNA (100 nmole each) and with SOD1-GFP or SOD1 G93A -GFP expression vectors. After incubation for 48 h, the cells were collected. Cell lysates were loaded to a non-reducing gel followed by western blot analysis with indicated antibodies. The data in Fig. 8d were derived from Fig. 8c in which only the density of protein aggregates (excluding the heavy band of the 53 kDa monomer) was quantified. Data in Fig. 8e were derived from Fig. 8c in which the relative levels of ATG7 and LC3 lipidation were analyzed. * p
    Rapamycin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem rapamycin
    Longer <t>rapamycin</t> infusion suppressed mossy fiber sprouting more, but the effect reversed after infusion ceased. Timm stained dentate gyrus after 2 months of continuous infusion with 10 mM rapamycin ( A ) and contralateral noninfused hippocampus ( B ). The
    Rapamycin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 415 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology rapamycin
    Inhibition of autophagy promotes AA, BSO and CCl 4 induced mitochondrial ROS formation. (A) (a), E47 cells were treated with AA, BSO or CCl 4 in the absence or presence of 3-MA or <t>rapamycin</t> for 48 h, and cells were then incubated with 5 µM MitoSox for 30 min. The red-stained mitochondria were observed under a fluorescence microscope (magnification ×100). (b), Cells were also re-suspended in 1×PBS and the intensity of fluorescence was determined in a Perkin-Elmer fluorescence spectrophotometer at Ex510/Em580 nm. ⁎ P
    Rapamycin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 425 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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    98
    InvivoGen rapamycin
    <t>Sirolimus</t> prevents weight gain and decreases fat mass of rats fed a HF diet
    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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    93
    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: 93/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, 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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    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:

    A77 1726 induces SOD1 G93A co-localization with autophagosomes. RFP-LC3 stably transfected NSC34 cells were transiently transfected with SOD1-GFP ( a ) or SOD1 G93A -GFP ( b ) expression vectors. After incubation for 40 h, the cells were treated with DMSO (0.2%), A77 1726 (200 μM) or rapamycin (50 nM) ( a ) for 24 h. The cells were fixed and examined under a confocal microscope for the localization of autophagosomes (RFP-LC3) and for SOD1-GFP or SOD1 G93A -GFP protein aggregates. c–e NSC34 cells were transfected with control or ATG7 siRNA (100 nmole each) and with SOD1-GFP or SOD1 G93A -GFP expression vectors. After incubation for 48 h, the cells were collected. Cell lysates were loaded to a non-reducing gel followed by western blot analysis with indicated antibodies. The data in Fig. 8d were derived from Fig. 8c in which only the density of protein aggregates (excluding the heavy band of the 53 kDa monomer) was quantified. Data in Fig. 8e were derived from Fig. 8c in which the relative levels of ATG7 and LC3 lipidation were analyzed. * p

    Journal: Cell Death & Disease

    Article Title: Inhibition of p70 S6 kinase activity by A77 1726 induces autophagy and enhances the degradation of superoxide dismutase 1 (SOD1) protein aggregates

    doi: 10.1038/s41419-018-0441-0

    Figure Lengend Snippet: A77 1726 induces SOD1 G93A co-localization with autophagosomes. RFP-LC3 stably transfected NSC34 cells were transiently transfected with SOD1-GFP ( a ) or SOD1 G93A -GFP ( b ) expression vectors. After incubation for 40 h, the cells were treated with DMSO (0.2%), A77 1726 (200 μM) or rapamycin (50 nM) ( a ) for 24 h. The cells were fixed and examined under a confocal microscope for the localization of autophagosomes (RFP-LC3) and for SOD1-GFP or SOD1 G93A -GFP protein aggregates. c–e NSC34 cells were transfected with control or ATG7 siRNA (100 nmole each) and with SOD1-GFP or SOD1 G93A -GFP expression vectors. After incubation for 48 h, the cells were collected. Cell lysates were loaded to a non-reducing gel followed by western blot analysis with indicated antibodies. The data in Fig. 8d were derived from Fig. 8c in which only the density of protein aggregates (excluding the heavy band of the 53 kDa monomer) was quantified. Data in Fig. 8e were derived from Fig. 8c in which the relative levels of ATG7 and LC3 lipidation were analyzed. * p

    Article Snippet: Rapamycin was purchased from Cayman Laboratories (Ann Arbor, MI).

    Techniques: Stable Transfection, Transfection, Expressing, Incubation, Microscopy, Western Blot, Derivative Assay

    A77 1726 blocks the formation of SOD1 G93A protein aggregates. a , b NSC34 cells transiently transfected with SOD1-GFP or SOD1 G93A -GFP expression vectors were treated as described in “Materials and Methods” section. The cells were examined under a confocal microscope for SOD1-GFP or SOD1 G93A -GFP expression ( a , b ) and quantified for the fluorescence intensity in a plate reader ( c ). The results represent the mean ± SD from one experiment in triplicate. The experiments were repeated twice with similar results. d The anti-proliferative effect of A77 1726 on NSC34 cells. Untransfected NSC34 cells or the cells transfected with the SOD1-GFP or SOD1 G93A -GFP expression vector were seeded in a 96-well plate (5000 cells per well) and incubated in the absence or presence of A77 1726 (200 μM) or rapamycin (50 nM) for 24 h. Cell proliferation was analyzed by an ATP-based Cell-Glo assay. The data are the mean ± SD of the triplicate from one representative of two experiments with similar results. * p

    Journal: Cell Death & Disease

    Article Title: Inhibition of p70 S6 kinase activity by A77 1726 induces autophagy and enhances the degradation of superoxide dismutase 1 (SOD1) protein aggregates

    doi: 10.1038/s41419-018-0441-0

    Figure Lengend Snippet: A77 1726 blocks the formation of SOD1 G93A protein aggregates. a , b NSC34 cells transiently transfected with SOD1-GFP or SOD1 G93A -GFP expression vectors were treated as described in “Materials and Methods” section. The cells were examined under a confocal microscope for SOD1-GFP or SOD1 G93A -GFP expression ( a , b ) and quantified for the fluorescence intensity in a plate reader ( c ). The results represent the mean ± SD from one experiment in triplicate. The experiments were repeated twice with similar results. d The anti-proliferative effect of A77 1726 on NSC34 cells. Untransfected NSC34 cells or the cells transfected with the SOD1-GFP or SOD1 G93A -GFP expression vector were seeded in a 96-well plate (5000 cells per well) and incubated in the absence or presence of A77 1726 (200 μM) or rapamycin (50 nM) for 24 h. Cell proliferation was analyzed by an ATP-based Cell-Glo assay. The data are the mean ± SD of the triplicate from one representative of two experiments with similar results. * p

    Article Snippet: Rapamycin was purchased from Cayman Laboratories (Ann Arbor, MI).

    Techniques: Transfection, Expressing, Microscopy, Fluorescence, Plasmid Preparation, Incubation, Glo Assay

    Evidence that A77 1726 induces SOD1 protein degradation. a NSC34 cells seeded in 60 mm dishes were transfected the SOD1-GFP or SOD1 G93A -GFP expression vectors and treated with A77 1726 (200 μM) or rapamycin (50 nM). Single-cell suspensions were analyzed for GFP expression in a flow cytometer. The fluorescence intensity was analyzed by using FlowJo software. The results represent the mean ± SD from three independent experiments. * p

    Journal: Cell Death & Disease

    Article Title: Inhibition of p70 S6 kinase activity by A77 1726 induces autophagy and enhances the degradation of superoxide dismutase 1 (SOD1) protein aggregates

    doi: 10.1038/s41419-018-0441-0

    Figure Lengend Snippet: Evidence that A77 1726 induces SOD1 protein degradation. a NSC34 cells seeded in 60 mm dishes were transfected the SOD1-GFP or SOD1 G93A -GFP expression vectors and treated with A77 1726 (200 μM) or rapamycin (50 nM). Single-cell suspensions were analyzed for GFP expression in a flow cytometer. The fluorescence intensity was analyzed by using FlowJo software. The results represent the mean ± SD from three independent experiments. * p

    Article Snippet: Rapamycin was purchased from Cayman Laboratories (Ann Arbor, MI).

    Techniques: Transfection, Expressing, Flow Cytometry, Cytometry, Fluorescence, Software

    The effect of A77 1726 on the feedback activation of the PI-3 kinase pathway and autophagy. a – f The effect of A77 1726 on the feedback activation of the PI-3 kinase pathway and LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of A77 1726 for 16 h ( a , c , d ) or were incubated in the presence of A77 1726 (200 μM) for the indicated time ( b , e , f ). Rapamycin (50 nM) was included as a positive control ( a , d , c ). Cell lysates were analyzed for the feedback activation of the PI-3 kinase pathway ( a , b ) or for LC3-II lipidation ( c , d ) by western blot with the indicated antibodies. g , h The effect of bafilomycin and colchicine on LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of A77 1726 (200 μM) minus or plus bafilomycin (100 nM) ( g , j ) or colchicine (5 μM) ( h , j ) for 16 h. Cell lysates were analyzed for LC3 and actin expression by western blot. i , j Inability of uridine to block A77 1726-induced LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of A77 1726 (200 μM) minus or plus uridine (200 μM) for 16 h. Cell lysates were analyzed for LC3-II lipidation and actin expression by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs ( c , f , j ). LC3 lipidation was analyzed by comparing the density of LC3-II with β-actin. The data in Fig. 1c, f, j and the remaining Image-J-derived data in all other figures are the mean ± SD from three experiments . k , l NSC34 cells were transfected with the expression vector pmLC3-RFP. The cells were left untreated or treated with A77 1726 (200 μM) or rapamycin (50 nM) for 16 h. Autophagosomes were visualized under a confocal microscope ( k ). The puncta of autophagosomes were counted under a fluorescent microscope and plotted in a bar graph with statistical analysis ( l ). * p

    Journal: Cell Death & Disease

    Article Title: Inhibition of p70 S6 kinase activity by A77 1726 induces autophagy and enhances the degradation of superoxide dismutase 1 (SOD1) protein aggregates

    doi: 10.1038/s41419-018-0441-0

    Figure Lengend Snippet: The effect of A77 1726 on the feedback activation of the PI-3 kinase pathway and autophagy. a – f The effect of A77 1726 on the feedback activation of the PI-3 kinase pathway and LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of A77 1726 for 16 h ( a , c , d ) or were incubated in the presence of A77 1726 (200 μM) for the indicated time ( b , e , f ). Rapamycin (50 nM) was included as a positive control ( a , d , c ). Cell lysates were analyzed for the feedback activation of the PI-3 kinase pathway ( a , b ) or for LC3-II lipidation ( c , d ) by western blot with the indicated antibodies. g , h The effect of bafilomycin and colchicine on LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of A77 1726 (200 μM) minus or plus bafilomycin (100 nM) ( g , j ) or colchicine (5 μM) ( h , j ) for 16 h. Cell lysates were analyzed for LC3 and actin expression by western blot. i , j Inability of uridine to block A77 1726-induced LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of A77 1726 (200 μM) minus or plus uridine (200 μM) for 16 h. Cell lysates were analyzed for LC3-II lipidation and actin expression by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs ( c , f , j ). LC3 lipidation was analyzed by comparing the density of LC3-II with β-actin. The data in Fig. 1c, f, j and the remaining Image-J-derived data in all other figures are the mean ± SD from three experiments . k , l NSC34 cells were transfected with the expression vector pmLC3-RFP. The cells were left untreated or treated with A77 1726 (200 μM) or rapamycin (50 nM) for 16 h. Autophagosomes were visualized under a confocal microscope ( k ). The puncta of autophagosomes were counted under a fluorescent microscope and plotted in a bar graph with statistical analysis ( l ). * p

    Article Snippet: Rapamycin was purchased from Cayman Laboratories (Ann Arbor, MI).

    Techniques: Activation Assay, Incubation, Positive Control, Western Blot, Expressing, Blocking Assay, Software, Derivative Assay, Transfection, Plasmid Preparation, Microscopy

    Longer rapamycin infusion suppressed mossy fiber sprouting more, but the effect reversed after infusion ceased. Timm stained dentate gyrus after 2 months of continuous infusion with 10 mM rapamycin ( A ) and contralateral noninfused hippocampus ( B ). The

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Inhibition of the Mammalian Target of Rapamycin Signaling Pathway Suppresses Dentate Granule Cell Axon Sprouting in a Rodent Model of Temporal Lobe Epilepsy

    doi: 10.1523/JNEUROSCI.4179-08.2009

    Figure Lengend Snippet: Longer rapamycin infusion suppressed mossy fiber sprouting more, but the effect reversed after infusion ceased. Timm stained dentate gyrus after 2 months of continuous infusion with 10 mM rapamycin ( A ) and contralateral noninfused hippocampus ( B ). The

    Article Snippet: Osmotic pumps and cannulae were implanted to focally and continuously deliver rapamycin (Alexis Biochemicals or LC Laboratories) to the dorsal, left dentate gyrus.

    Techniques: Staining

    Density of staining within Timm-positive contours was not more intense in rapamycin-infused hippocampi. The average optical density (0, no tissue in light path; 1, microscope light source turned off) within Timm-positive contours in three sections within

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Inhibition of the Mammalian Target of Rapamycin Signaling Pathway Suppresses Dentate Granule Cell Axon Sprouting in a Rodent Model of Temporal Lobe Epilepsy

    doi: 10.1523/JNEUROSCI.4179-08.2009

    Figure Lengend Snippet: Density of staining within Timm-positive contours was not more intense in rapamycin-infused hippocampi. The average optical density (0, no tissue in light path; 1, microscope light source turned off) within Timm-positive contours in three sections within

    Article Snippet: Osmotic pumps and cannulae were implanted to focally and continuously deliver rapamycin (Alexis Biochemicals or LC Laboratories) to the dorsal, left dentate gyrus.

    Techniques: Staining, Microscopy

    Rapamycin infusion did not protect hilar neurons from status epilepticus-induced loss. Nissl stained dentate gyrus after 1 month infusion with 10 mM rapamycin ( A ) and contralateral noninfused hippocampus ( B ). The asterisk indicates the infusion site.

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Inhibition of the Mammalian Target of Rapamycin Signaling Pathway Suppresses Dentate Granule Cell Axon Sprouting in a Rodent Model of Temporal Lobe Epilepsy

    doi: 10.1523/JNEUROSCI.4179-08.2009

    Figure Lengend Snippet: Rapamycin infusion did not protect hilar neurons from status epilepticus-induced loss. Nissl stained dentate gyrus after 1 month infusion with 10 mM rapamycin ( A ) and contralateral noninfused hippocampus ( B ). The asterisk indicates the infusion site.

    Article Snippet: Osmotic pumps and cannulae were implanted to focally and continuously deliver rapamycin (Alexis Biochemicals or LC Laboratories) to the dorsal, left dentate gyrus.

    Techniques: Staining

    The mTOR signaling pathway in hippocampus was activated by status epilepticus and inhibited by prolonged infusion of rapamycin. Ribosomal protein S6 is phosphorylated by the mTOR signaling pathway. Ratios of phospho-S6 to total S6 were measured by Western

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Inhibition of the Mammalian Target of Rapamycin Signaling Pathway Suppresses Dentate Granule Cell Axon Sprouting in a Rodent Model of Temporal Lobe Epilepsy

    doi: 10.1523/JNEUROSCI.4179-08.2009

    Figure Lengend Snippet: The mTOR signaling pathway in hippocampus was activated by status epilepticus and inhibited by prolonged infusion of rapamycin. Ribosomal protein S6 is phosphorylated by the mTOR signaling pathway. Ratios of phospho-S6 to total S6 were measured by Western

    Article Snippet: Osmotic pumps and cannulae were implanted to focally and continuously deliver rapamycin (Alexis Biochemicals or LC Laboratories) to the dorsal, left dentate gyrus.

    Techniques: Western Blot

    Fluorescence verified delivery of osmotic pump contents to infused but not contralateral dentate gyrus. A , Left, dorsal dentate gyrus of a rat infused for 1 month with 0.01 mM rapamycin dissolved in vehicle containing 0.1% fluorescein. The asterisk indicates

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Inhibition of the Mammalian Target of Rapamycin Signaling Pathway Suppresses Dentate Granule Cell Axon Sprouting in a Rodent Model of Temporal Lobe Epilepsy

    doi: 10.1523/JNEUROSCI.4179-08.2009

    Figure Lengend Snippet: Fluorescence verified delivery of osmotic pump contents to infused but not contralateral dentate gyrus. A , Left, dorsal dentate gyrus of a rat infused for 1 month with 0.01 mM rapamycin dissolved in vehicle containing 0.1% fluorescein. The asterisk indicates

    Article Snippet: Osmotic pumps and cannulae were implanted to focally and continuously deliver rapamycin (Alexis Biochemicals or LC Laboratories) to the dorsal, left dentate gyrus.

    Techniques: Fluorescence

    Prolonged, 1 month infusion of rapamycin reduced aberrant Timm staining. Timm stained dentate gyrus in vehicle-infused ( A ) and contralateral noninfused ( B ) hippocampus and in 10 mM rapamycin-infused ( C ) and contralateral noninfused ( D ) hippocampus. Asterisks

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Inhibition of the Mammalian Target of Rapamycin Signaling Pathway Suppresses Dentate Granule Cell Axon Sprouting in a Rodent Model of Temporal Lobe Epilepsy

    doi: 10.1523/JNEUROSCI.4179-08.2009

    Figure Lengend Snippet: Prolonged, 1 month infusion of rapamycin reduced aberrant Timm staining. Timm stained dentate gyrus in vehicle-infused ( A ) and contralateral noninfused ( B ) hippocampus and in 10 mM rapamycin-infused ( C ) and contralateral noninfused ( D ) hippocampus. Asterisks

    Article Snippet: Osmotic pumps and cannulae were implanted to focally and continuously deliver rapamycin (Alexis Biochemicals or LC Laboratories) to the dorsal, left dentate gyrus.

    Techniques: Staining

    Rapamycin infusion did not reverse mossy fiber sprouting after it had developed. Timm stained dentate gyrus after 1 month infusion with 10 mM rapamycin, beginning 2 months after status epilepticus ( A ) and contralateral noninfused hippocampus ( B ). The

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Inhibition of the Mammalian Target of Rapamycin Signaling Pathway Suppresses Dentate Granule Cell Axon Sprouting in a Rodent Model of Temporal Lobe Epilepsy

    doi: 10.1523/JNEUROSCI.4179-08.2009

    Figure Lengend Snippet: Rapamycin infusion did not reverse mossy fiber sprouting after it had developed. Timm stained dentate gyrus after 1 month infusion with 10 mM rapamycin, beginning 2 months after status epilepticus ( A ) and contralateral noninfused hippocampus ( B ). The

    Article Snippet: Osmotic pumps and cannulae were implanted to focally and continuously deliver rapamycin (Alexis Biochemicals or LC Laboratories) to the dorsal, left dentate gyrus.

    Techniques: Staining

    Inhibition of autophagy promotes AA, BSO and CCl 4 induced mitochondrial ROS formation. (A) (a), E47 cells were treated with AA, BSO or CCl 4 in the absence or presence of 3-MA or rapamycin for 48 h, and cells were then incubated with 5 µM MitoSox for 30 min. The red-stained mitochondria were observed under a fluorescence microscope (magnification ×100). (b), Cells were also re-suspended in 1×PBS and the intensity of fluorescence was determined in a Perkin-Elmer fluorescence spectrophotometer at Ex510/Em580 nm. ⁎ P

    Journal: Redox Biology

    Article Title: Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

    doi: 10.1016/j.redox.2013.10.008

    Figure Lengend Snippet: Inhibition of autophagy promotes AA, BSO and CCl 4 induced mitochondrial ROS formation. (A) (a), E47 cells were treated with AA, BSO or CCl 4 in the absence or presence of 3-MA or rapamycin for 48 h, and cells were then incubated with 5 µM MitoSox for 30 min. The red-stained mitochondria were observed under a fluorescence microscope (magnification ×100). (b), Cells were also re-suspended in 1×PBS and the intensity of fluorescence was determined in a Perkin-Elmer fluorescence spectrophotometer at Ex510/Em580 nm. ⁎ P

    Article Snippet: Activation of p38 MAP kinase and JNK E47 cells were treated with 20 µM AA in the presence and absence of 3-MA or rapamycin for 6, 12, 24 and 48 h. Cell lysates were collected and immunoblots were carried out to detect levels of pp38 MAPK, p38 MAPK, pJNK and JNK (antibodies from Santa Cruz).

    Techniques: Inhibition, Incubation, Staining, Fluorescence, Microscopy, Spectrophotometry

    Inhibition of autophagy promotes AA activation of p38 MAPK and JNK. E47 cells were treated with 3-MA or rapamycin in the presence or absence of AA (45 µM) for 6, 12, 24 and 48 h. Cell lysates were prepared and immunoblots were carried out to determine the time course activation of p38 MAPK (A and B) or JNK (C and D). The ratio of pp38 MAPK/p38 MAPK is listed at the bottoms of blots A and B ( ⁎ P

    Journal: Redox Biology

    Article Title: Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

    doi: 10.1016/j.redox.2013.10.008

    Figure Lengend Snippet: Inhibition of autophagy promotes AA activation of p38 MAPK and JNK. E47 cells were treated with 3-MA or rapamycin in the presence or absence of AA (45 µM) for 6, 12, 24 and 48 h. Cell lysates were prepared and immunoblots were carried out to determine the time course activation of p38 MAPK (A and B) or JNK (C and D). The ratio of pp38 MAPK/p38 MAPK is listed at the bottoms of blots A and B ( ⁎ P

    Article Snippet: Activation of p38 MAP kinase and JNK E47 cells were treated with 20 µM AA in the presence and absence of 3-MA or rapamycin for 6, 12, 24 and 48 h. Cell lysates were collected and immunoblots were carried out to detect levels of pp38 MAPK, p38 MAPK, pJNK and JNK (antibodies from Santa Cruz).

    Techniques: Inhibition, Activation Assay, Western Blot

    Autophagy modulates AA, BSO and CCl 4 induced mitochondrial dysfunction. E47 cells were treated with AA, BSO or CCl 4 with or without 3-MA or rapamycin for 48 h and mitochondria were freshly prepared. Mitochondrial membrane swelling (A–C) was evaluated by determining the decrease in absorbance at 540 nm after the addition of 100 µM CaCl 2 . (A) AA; (B) BSO; (C) CCl 4 ; (D) Cellular ATP levels ( ⁎ P

    Journal: Redox Biology

    Article Title: Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

    doi: 10.1016/j.redox.2013.10.008

    Figure Lengend Snippet: Autophagy modulates AA, BSO and CCl 4 induced mitochondrial dysfunction. E47 cells were treated with AA, BSO or CCl 4 with or without 3-MA or rapamycin for 48 h and mitochondria were freshly prepared. Mitochondrial membrane swelling (A–C) was evaluated by determining the decrease in absorbance at 540 nm after the addition of 100 µM CaCl 2 . (A) AA; (B) BSO; (C) CCl 4 ; (D) Cellular ATP levels ( ⁎ P

    Article Snippet: Activation of p38 MAP kinase and JNK E47 cells were treated with 20 µM AA in the presence and absence of 3-MA or rapamycin for 6, 12, 24 and 48 h. Cell lysates were collected and immunoblots were carried out to detect levels of pp38 MAPK, p38 MAPK, pJNK and JNK (antibodies from Santa Cruz).

    Techniques:

    Autophagy protects against CYP2E1-mediated AA, CCl 4 and BSO cell toxicity. HepG2 E47 and C34 cells were treated with AA (20 µM), CCl 4 (300 ug/ml) or BSO (300 µM) for 48 h in the presence and absence of 3-MA (2.5 mM) or rapamycin (0.2 µg/ml). (A) Cell viability was determined by a MTT assay (* P

    Journal: Redox Biology

    Article Title: Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

    doi: 10.1016/j.redox.2013.10.008

    Figure Lengend Snippet: Autophagy protects against CYP2E1-mediated AA, CCl 4 and BSO cell toxicity. HepG2 E47 and C34 cells were treated with AA (20 µM), CCl 4 (300 ug/ml) or BSO (300 µM) for 48 h in the presence and absence of 3-MA (2.5 mM) or rapamycin (0.2 µg/ml). (A) Cell viability was determined by a MTT assay (* P

    Article Snippet: Activation of p38 MAP kinase and JNK E47 cells were treated with 20 µM AA in the presence and absence of 3-MA or rapamycin for 6, 12, 24 and 48 h. Cell lysates were collected and immunoblots were carried out to detect levels of pp38 MAPK, p38 MAPK, pJNK and JNK (antibodies from Santa Cruz).

    Techniques: MTT Assay

    Knockdown of Atg 7 promotes AA induced ROS formation and cytotoxicity. (A) Transfection of E47 cells with Atg 7 siRNA but not control siRNA for 48 h lowers levels of Atg 7. (B) E47 cells were transfected with Atg 7 siRNA or control siRNA for 48 h. Cells were then treated with AA (20 µM) or rapamycin for an additional 48 h and cell viability determined by a MTT assay. ( ⁎ P

    Journal: Redox Biology

    Article Title: Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

    doi: 10.1016/j.redox.2013.10.008

    Figure Lengend Snippet: Knockdown of Atg 7 promotes AA induced ROS formation and cytotoxicity. (A) Transfection of E47 cells with Atg 7 siRNA but not control siRNA for 48 h lowers levels of Atg 7. (B) E47 cells were transfected with Atg 7 siRNA or control siRNA for 48 h. Cells were then treated with AA (20 µM) or rapamycin for an additional 48 h and cell viability determined by a MTT assay. ( ⁎ P

    Article Snippet: Activation of p38 MAP kinase and JNK E47 cells were treated with 20 µM AA in the presence and absence of 3-MA or rapamycin for 6, 12, 24 and 48 h. Cell lysates were collected and immunoblots were carried out to detect levels of pp38 MAPK, p38 MAPK, pJNK and JNK (antibodies from Santa Cruz).

    Techniques: Transfection, MTT Assay

    Pharmacological inhibition of autophagy enhances AA, BSO or CCl 4 induced ROS stress. E47 cells were treated with AA, BSO or CCl 4 in the presence or absence of 3-MA or rapamycin for 48 h. (A) Lipid peroxidation was determined by measuring the formation of TBARs products. (B) GSH levels were determined with GSH reductase. ( P

    Journal: Redox Biology

    Article Title: Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

    doi: 10.1016/j.redox.2013.10.008

    Figure Lengend Snippet: Pharmacological inhibition of autophagy enhances AA, BSO or CCl 4 induced ROS stress. E47 cells were treated with AA, BSO or CCl 4 in the presence or absence of 3-MA or rapamycin for 48 h. (A) Lipid peroxidation was determined by measuring the formation of TBARs products. (B) GSH levels were determined with GSH reductase. ( P

    Article Snippet: Activation of p38 MAP kinase and JNK E47 cells were treated with 20 µM AA in the presence and absence of 3-MA or rapamycin for 6, 12, 24 and 48 h. Cell lysates were collected and immunoblots were carried out to detect levels of pp38 MAPK, p38 MAPK, pJNK and JNK (antibodies from Santa Cruz).

    Techniques: Inhibition

    Effects of AA, BSO or CCl 4 on autophagy-related proteins and CYP2E1. E47 cells were treated with AA, BSO or CCl 4 in the presence or absence of 3-MA or rapamycin for 48 h as described in the legend to Fig. 1 . (A) LC3 levels were evaluated by immunoblots and the LC3-II/LC3-I ratio calculated (⁎, # P

    Journal: Redox Biology

    Article Title: Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

    doi: 10.1016/j.redox.2013.10.008

    Figure Lengend Snippet: Effects of AA, BSO or CCl 4 on autophagy-related proteins and CYP2E1. E47 cells were treated with AA, BSO or CCl 4 in the presence or absence of 3-MA or rapamycin for 48 h as described in the legend to Fig. 1 . (A) LC3 levels were evaluated by immunoblots and the LC3-II/LC3-I ratio calculated (⁎, # P

    Article Snippet: Activation of p38 MAP kinase and JNK E47 cells were treated with 20 µM AA in the presence and absence of 3-MA or rapamycin for 6, 12, 24 and 48 h. Cell lysates were collected and immunoblots were carried out to detect levels of pp38 MAPK, p38 MAPK, pJNK and JNK (antibodies from Santa Cruz).

    Techniques: Western Blot

    A p38MAPK inhibitor and a JNK inhibitor decrease 3-MA enhanced AA cytotoxicity. E47 cells were treated with 45 µM AA plus 3-MA or rapamycin in the presence or absence of SB203580 (5 µM) or SP600125 (2.5 µM) for 24 and 48 h. (A): cell viability was determined by a MTT assay (⁎, # P

    Journal: Redox Biology

    Article Title: Inhibition of autophagy promotes CYP2E1-dependent toxicity in HepG2 cells via elevated oxidative stress, mitochondria dysfunction and activation of p38 and JNK MAPK

    doi: 10.1016/j.redox.2013.10.008

    Figure Lengend Snippet: A p38MAPK inhibitor and a JNK inhibitor decrease 3-MA enhanced AA cytotoxicity. E47 cells were treated with 45 µM AA plus 3-MA or rapamycin in the presence or absence of SB203580 (5 µM) or SP600125 (2.5 µM) for 24 and 48 h. (A): cell viability was determined by a MTT assay (⁎, # P

    Article Snippet: Activation of p38 MAP kinase and JNK E47 cells were treated with 20 µM AA in the presence and absence of 3-MA or rapamycin for 6, 12, 24 and 48 h. Cell lysates were collected and immunoblots were carried out to detect levels of pp38 MAPK, p38 MAPK, pJNK and JNK (antibodies from Santa Cruz).

    Techniques: MTT Assay

    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