rapamycin  (Millipore)


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  • 99
    Name:
    Rapamycin from Streptomyces hygroscopicus
    Description:
    Chemical structure macrolide
    Catalog Number:
    r0395
    Price:
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    Structured Review

    Millipore rapamycin
    Rapamycin from Streptomyces hygroscopicus
    Chemical structure macrolide
    https://www.bioz.com/result/rapamycin/product/Millipore
    Average 99 stars, based on 458 article reviews
    Price from $9.99 to $1999.99
    rapamycin - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury"

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

    Journal: Autophagy

    doi: 10.4161/auto.18980

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

    Techniques Used: Isolation, Mouse Assay, Cell Culture

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

    Techniques Used: Cell Culture, Western Blot

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

    Techniques Used:

    2) Product Images from "THR1 mediates GCN4 and CDC4 to link morphogenesis with nutrient sensing and the stress response in Candida albicans"

    Article Title: THR1 mediates GCN4 and CDC4 to link morphogenesis with nutrient sensing and the stress response in Candida albicans

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3930

    Cells without GCN4 rescue those without THR1 that are sensitive to activation of the TOR pathway by Rapa or 3-AT. The growth of C. albicans strains assayed using a spotting assay on limited nutrient conditions. Wild-type, null mutants of gcn4, thr1, gcn4 thr1 were grown on (A) YPD with or without homoserine at 30°C for 2 days, with (B) on YPD with or without indicated Rapa at 30°C for 2 days or SC with or without indicated 3-AT plates at 30°C for 3 days, or (C) on SD plates at 30°C for 3 days with or without different concentration of Asp, Thr, or their combination: 1× Asp, 10× Asp, 1× Thr, 10× Thr, 1× Asp + 1× Thr, and 10× Asp + 10× Thr. 1×, 80 ng ml −1 ; 10×, 800 ng ml −1 ; SC, synthetic complete medium; SD, synthetic defined medium lacking amino acids; Rapa, rapamycin; 3-AT, 3-amino-1,2,4-triazole; Asp, aspartate; Thr, threonine.
    Figure Legend Snippet: Cells without GCN4 rescue those without THR1 that are sensitive to activation of the TOR pathway by Rapa or 3-AT. The growth of C. albicans strains assayed using a spotting assay on limited nutrient conditions. Wild-type, null mutants of gcn4, thr1, gcn4 thr1 were grown on (A) YPD with or without homoserine at 30°C for 2 days, with (B) on YPD with or without indicated Rapa at 30°C for 2 days or SC with or without indicated 3-AT plates at 30°C for 3 days, or (C) on SD plates at 30°C for 3 days with or without different concentration of Asp, Thr, or their combination: 1× Asp, 10× Asp, 1× Thr, 10× Thr, 1× Asp + 1× Thr, and 10× Asp + 10× Thr. 1×, 80 ng ml −1 ; 10×, 800 ng ml −1 ; SC, synthetic complete medium; SD, synthetic defined medium lacking amino acids; Rapa, rapamycin; 3-AT, 3-amino-1,2,4-triazole; Asp, aspartate; Thr, threonine.

    Techniques Used: Activation Assay, Spotting Assay, Concentration Assay

    Nutrient limitation induces the expression of GCN4 and THR1 . (A) RT-PCR analyses of the mRNA levels of GCN4 and THR1 were performed following 0, 1, 3 and 6 h of growth in YPD with or without 10 nM Rapa (upper panel) or 10 mM 3-AT (lower panel). (B) RT-PCR analyses of mRNA levels of GCN4 and THR1 were performed following 3 h of growth in YPD in the presence of absence of 10 and 40 nM Rapa (upper panel) or 10 and 40 mM 3-AT (lower panel). The mRNA level of ACT1 was used as a loading control. Rapa, rapamycin; 3-AT, 3-amino-1,2,4-triazole; RT-PCR, reverse transcription-polymerase chain reaction.
    Figure Legend Snippet: Nutrient limitation induces the expression of GCN4 and THR1 . (A) RT-PCR analyses of the mRNA levels of GCN4 and THR1 were performed following 0, 1, 3 and 6 h of growth in YPD with or without 10 nM Rapa (upper panel) or 10 mM 3-AT (lower panel). (B) RT-PCR analyses of mRNA levels of GCN4 and THR1 were performed following 3 h of growth in YPD in the presence of absence of 10 and 40 nM Rapa (upper panel) or 10 and 40 mM 3-AT (lower panel). The mRNA level of ACT1 was used as a loading control. Rapa, rapamycin; 3-AT, 3-amino-1,2,4-triazole; RT-PCR, reverse transcription-polymerase chain reaction.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    3) Product Images from "Autophagy is a protective response to ethanol neurotoxicity"

    Article Title: Autophagy is a protective response to ethanol neurotoxicity

    Journal: Autophagy

    doi: 10.4161/auto.21376

    Figure 6. Effect of ethanol, rapamycin, wortmannin, catalase or BECN1 shRNA on the viability of SH-SY5Y cells. SH-SY5Y cells were treated with ethanol (0 or 0.8%) with/without rapamycin (10 nM), catalase (10,000 U/ml) or wortmannin (10 µM) for 48 h ( A ). In some experimental groups, cells were treated with a BECN1 shRNA to downregulate the expression of BECN1 ( B ). Cell viability was determined by MTT assay as described under Materials and Methods. The data represent the mean and SEM of three replications. *p
    Figure Legend Snippet: Figure 6. Effect of ethanol, rapamycin, wortmannin, catalase or BECN1 shRNA on the viability of SH-SY5Y cells. SH-SY5Y cells were treated with ethanol (0 or 0.8%) with/without rapamycin (10 nM), catalase (10,000 U/ml) or wortmannin (10 µM) for 48 h ( A ). In some experimental groups, cells were treated with a BECN1 shRNA to downregulate the expression of BECN1 ( B ). Cell viability was determined by MTT assay as described under Materials and Methods. The data represent the mean and SEM of three replications. *p

    Techniques Used: shRNA, Expressing, MTT Assay

    Figure 5. Effect of ethanol on ROS generation in SH-SY5Y cells. SH-SY5Y cells were treated with ethanol (0 or 0.8%) with/without rapamycin (10 nM) or bafilomycin A 1 (10 nM) for 2 or 6 h. In some experimental groups, cells were treated with BECN1 shRNA to downregulate the expression of BECN1. ROS generation was measured as described under the Materials and Methods and relative amounts of ROS are presented. The data represent the mean and SEM of three replications. *p
    Figure Legend Snippet: Figure 5. Effect of ethanol on ROS generation in SH-SY5Y cells. SH-SY5Y cells were treated with ethanol (0 or 0.8%) with/without rapamycin (10 nM) or bafilomycin A 1 (10 nM) for 2 or 6 h. In some experimental groups, cells were treated with BECN1 shRNA to downregulate the expression of BECN1. ROS generation was measured as described under the Materials and Methods and relative amounts of ROS are presented. The data represent the mean and SEM of three replications. *p

    Techniques Used: shRNA, Expressing

    Figure 7. Effect of ethanol and rapamycin on caspase-3 activation in the developing brain. PD7 mice were injected with ethanol (0 or 5 g/kg) and/or rapamycin (10 mg/kg or 20 mg/kg, Rapa: 10 or 20). The expression of active CASP3/caspase-3 in the cerebral cortex was examined 8 h after the injection by immunohistochemistry as described under Materials and Methods. Scale bar: 100 μm. The experiment was replicated three times.
    Figure Legend Snippet: Figure 7. Effect of ethanol and rapamycin on caspase-3 activation in the developing brain. PD7 mice were injected with ethanol (0 or 5 g/kg) and/or rapamycin (10 mg/kg or 20 mg/kg, Rapa: 10 or 20). The expression of active CASP3/caspase-3 in the cerebral cortex was examined 8 h after the injection by immunohistochemistry as described under Materials and Methods. Scale bar: 100 μm. The experiment was replicated three times.

    Techniques Used: Activation Assay, Mouse Assay, Injection, Expressing, Immunohistochemistry

    Figure 2. Effect of ethanol on autophagic flux in SH-SY5Y cells. SH-SY5Y cells were treated with ethanol (0 or 0.8%) in the presence/absence of wortmannin (Wort: 10 µM), bafilomycin A 1 (Baf: 10 nM), rapamycin (Rap: 10 nM) or BECN1 shRNA (BECN1). The protein samples were collected 8 h after the treatment. The levels of LC3 ( A ) and SQSTM1 ( B ) were examined with immunoblotting (top panel). The experiment was replicated three times. Relative levels of LC3-II or SQSTM1 were determined by densitometry and normalized to actin levels (bottom panel). *p
    Figure Legend Snippet: Figure 2. Effect of ethanol on autophagic flux in SH-SY5Y cells. SH-SY5Y cells were treated with ethanol (0 or 0.8%) in the presence/absence of wortmannin (Wort: 10 µM), bafilomycin A 1 (Baf: 10 nM), rapamycin (Rap: 10 nM) or BECN1 shRNA (BECN1). The protein samples were collected 8 h after the treatment. The levels of LC3 ( A ) and SQSTM1 ( B ) were examined with immunoblotting (top panel). The experiment was replicated three times. Relative levels of LC3-II or SQSTM1 were determined by densitometry and normalized to actin levels (bottom panel). *p

    Techniques Used: shRNA

    4) Product Images from "Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus"

    Article Title: Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus

    Journal: Autophagy

    doi: 10.4161/auto.23465

    Figure 8. IBV does not inhibit autophagy induced by HBSS starvation or rapamycin. CK cells were transduced with rAd-EGFP-Av-LC3B and 24 h later, either mock-infected (open bars) or infected with IBV Beau-R (closed bars). At 24 hpi, cells were
    Figure Legend Snippet: Figure 8. IBV does not inhibit autophagy induced by HBSS starvation or rapamycin. CK cells were transduced with rAd-EGFP-Av-LC3B and 24 h later, either mock-infected (open bars) or infected with IBV Beau-R (closed bars). At 24 hpi, cells were

    Techniques Used: Transduction, Infection

    5) Product Images from "Autophagy activation prevents sevoflurane-induced neurotoxicity in H4 human neuroglioma cells"

    Article Title: Autophagy activation prevents sevoflurane-induced neurotoxicity in H4 human neuroglioma cells

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2016.6

    H4 cells were exposed to sevoflurane (0% or 4.1%) with/without rapamycin (Rap, 1 μmol/L) or 3-MA (5 mmol/L) for 6 h. The expression of CHOP and GRP78 was detected by Western blot analysis. The data represent the mean±SEM of three replications.
    Figure Legend Snippet: H4 cells were exposed to sevoflurane (0% or 4.1%) with/without rapamycin (Rap, 1 μmol/L) or 3-MA (5 mmol/L) for 6 h. The expression of CHOP and GRP78 was detected by Western blot analysis. The data represent the mean±SEM of three replications.

    Techniques Used: Expressing, Western Blot

    Activation of autophagy prevents sevoflurane-induced apoptosis in H4 cells. H4 cells were exposed to 0% or 4.1% sevoflurane with/without rapamycin (Rap, 1 μmol/L) or 3-MA (5 mmol/L) for 6 h. (A) Expression of LC3 detected by Western blotting.
    Figure Legend Snippet: Activation of autophagy prevents sevoflurane-induced apoptosis in H4 cells. H4 cells were exposed to 0% or 4.1% sevoflurane with/without rapamycin (Rap, 1 μmol/L) or 3-MA (5 mmol/L) for 6 h. (A) Expression of LC3 detected by Western blotting.

    Techniques Used: Activation Assay, Expressing, Western Blot

    6) Product Images from "ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2"

    Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

    Journal: Autophagy

    doi: 10.1080/15548627.2016.1217380

    Intestine-specific knockout of Abhd5 promotes tumorigenesis and aggressiveness of tumors in Apc Min/− mice in an autophagy-dependent manner. (A, B) Representative immunostaining images of expression of proliferation marker MKI67, apoptosis marker TUNEL, autophagy marker LC3 and SQSTM1 in the distal ilia of 100-d-old male mice with or without intestinal knockout of Abhd5 . Control, Abhd5 +/+/Cre+ ; ABHD5 knckout, Abhd5 f/f/Cre+ . (C) Western blots of LC3 and SQSTM1 in the entire small intestine of 100-d-old male mice. Control, Abhd5 +/+/Cre+ ; Abhd5 knockout, Abhd5 f/f/Cre+ . (D) Representative images of Swiss-role H E sections of the entire small intestine tissue of 100-d-old male and female Apc Min/− mice. Control, Apc Min/− Abhd5 +/+/Cre+ ; Abhd5 knockout, Apc Min/− Abhd5 f/f/Cre+ , the intestinal tumors were circled as shown. (E) Apc Min/− Abhd5 +/+/Cre+ and Apc Min/− Abhd5 f/f/Cre+ mice were intraperitoneally administered rapamycin (3 mg/kg, once daily from the age of 30 d until the age of 100 d), and the tumor number and size in the entire small intestine were statistically analyzed. (F) Apc Min/− Abhd5 +/+/Cre+ and Apc Min/− Abhd5 f/f/Cre+ mice were intraperitoneally administered rapamycin (3 mg/kg, once daily, from the age of 30 d until the age of 100 days), and representative H E sections of tumors in the small intestine of control and Apc Min/− Abhd5 f/f/Cre+ male mice at 100 d of age. (G) Apc Min/− Abhd5 +/+/Cre+ and Apc Min/− Abhd5 f/f/Cre+ mice were intraperitoneally administered rapamycin (3 mg/kg, once daily, from the age of 30 d until the age of 100 d), and representative immunostaining images of CDX2, a differentiation marker of intestinal epithelial cells, revealing the differentiation status of the intestinal epithelial cell of control and homozygous ( Apc Min/− Abhd5 f/f/Cre+ ) male mice at 100 d of age. (*, p
    Figure Legend Snippet: Intestine-specific knockout of Abhd5 promotes tumorigenesis and aggressiveness of tumors in Apc Min/− mice in an autophagy-dependent manner. (A, B) Representative immunostaining images of expression of proliferation marker MKI67, apoptosis marker TUNEL, autophagy marker LC3 and SQSTM1 in the distal ilia of 100-d-old male mice with or without intestinal knockout of Abhd5 . Control, Abhd5 +/+/Cre+ ; ABHD5 knckout, Abhd5 f/f/Cre+ . (C) Western blots of LC3 and SQSTM1 in the entire small intestine of 100-d-old male mice. Control, Abhd5 +/+/Cre+ ; Abhd5 knockout, Abhd5 f/f/Cre+ . (D) Representative images of Swiss-role H E sections of the entire small intestine tissue of 100-d-old male and female Apc Min/− mice. Control, Apc Min/− Abhd5 +/+/Cre+ ; Abhd5 knockout, Apc Min/− Abhd5 f/f/Cre+ , the intestinal tumors were circled as shown. (E) Apc Min/− Abhd5 +/+/Cre+ and Apc Min/− Abhd5 f/f/Cre+ mice were intraperitoneally administered rapamycin (3 mg/kg, once daily from the age of 30 d until the age of 100 d), and the tumor number and size in the entire small intestine were statistically analyzed. (F) Apc Min/− Abhd5 +/+/Cre+ and Apc Min/− Abhd5 f/f/Cre+ mice were intraperitoneally administered rapamycin (3 mg/kg, once daily, from the age of 30 d until the age of 100 days), and representative H E sections of tumors in the small intestine of control and Apc Min/− Abhd5 f/f/Cre+ male mice at 100 d of age. (G) Apc Min/− Abhd5 +/+/Cre+ and Apc Min/− Abhd5 f/f/Cre+ mice were intraperitoneally administered rapamycin (3 mg/kg, once daily, from the age of 30 d until the age of 100 d), and representative immunostaining images of CDX2, a differentiation marker of intestinal epithelial cells, revealing the differentiation status of the intestinal epithelial cell of control and homozygous ( Apc Min/− Abhd5 f/f/Cre+ ) male mice at 100 d of age. (*, p

    Techniques Used: Knock-Out, Mouse Assay, Immunostaining, Expressing, Marker, TUNEL Assay, Western Blot

    ABHD5 competitively occupies the CASP3-dependent cleavage sites of BECN1 to protect BECN1 from being cleaved by CASP3. (A) Western blots of apoptosis-related proteins in whole ABHD5 -silenced and control CCD841CON cells 24 h after treatment with PBS, EBSS or EBSS+rapamycin (50 nM). (B) CRISPR/Cas9-mediated CASP3 knockout (KO) CCD841CON cells were subjected to ABHD5 knockdown and a follow-up challenge of EBSS. The cytosolic and mitochondrial fractions were analyzed by western blotting using anti-BECN1-C with COX4I as a loading control, and the nuclear fractions were analyzed by western blotting using anti-BECN1-N with histone 2A as a loading control. (C) A scheme representing BECN1. Sequence alignment of human BECN1 shows conservation of the CASP3 cleavage sites, DLFD 124 , TDVD 133 and DQLD 149 . The CASP3-dependent cleavage sites are indicated in bold. The BH3, coiled-coil domain (CCD) and evolutionarily conserved domain (ECD) are depicted. (D) In vitro binding assay with FLAG-tagged different domains of BECN1 and His-tagged CASP3 or ABHD5 as indicated. The indicated A, B or C domain structure of BECN1 is given in (C). (E) Representative immunofluorescent images of staining with the indicated antibodies are shown. ABHD5, BECN1 and CASP3 are shown as blue, red, and green, respectively. An overlay (purple) in control cells reveals an interaction between ABHD5 and BECN1, and an overlay (orange) in ABHD5 -silenced cells reveals an interaction between BECN1 and CASP3. (F) Immunoprecipitation of endogenous BECN1 with LC3 in ABHD5 -silenced and control CCD841CON cells.
    Figure Legend Snippet: ABHD5 competitively occupies the CASP3-dependent cleavage sites of BECN1 to protect BECN1 from being cleaved by CASP3. (A) Western blots of apoptosis-related proteins in whole ABHD5 -silenced and control CCD841CON cells 24 h after treatment with PBS, EBSS or EBSS+rapamycin (50 nM). (B) CRISPR/Cas9-mediated CASP3 knockout (KO) CCD841CON cells were subjected to ABHD5 knockdown and a follow-up challenge of EBSS. The cytosolic and mitochondrial fractions were analyzed by western blotting using anti-BECN1-C with COX4I as a loading control, and the nuclear fractions were analyzed by western blotting using anti-BECN1-N with histone 2A as a loading control. (C) A scheme representing BECN1. Sequence alignment of human BECN1 shows conservation of the CASP3 cleavage sites, DLFD 124 , TDVD 133 and DQLD 149 . The CASP3-dependent cleavage sites are indicated in bold. The BH3, coiled-coil domain (CCD) and evolutionarily conserved domain (ECD) are depicted. (D) In vitro binding assay with FLAG-tagged different domains of BECN1 and His-tagged CASP3 or ABHD5 as indicated. The indicated A, B or C domain structure of BECN1 is given in (C). (E) Representative immunofluorescent images of staining with the indicated antibodies are shown. ABHD5, BECN1 and CASP3 are shown as blue, red, and green, respectively. An overlay (purple) in control cells reveals an interaction between ABHD5 and BECN1, and an overlay (orange) in ABHD5 -silenced cells reveals an interaction between BECN1 and CASP3. (F) Immunoprecipitation of endogenous BECN1 with LC3 in ABHD5 -silenced and control CCD841CON cells.

    Techniques Used: Western Blot, CRISPR, Knock-Out, Sequencing, In Vitro, Binding Assay, Staining, Immunoprecipitation

    Loss of ABHD5 suppresses CASP-independent cell death induced by nutrition deprivation. (A) GSEA plot of autophagy, apoptosis and WNT signaling pathways between ABHD5 high and ABHD5 low subgroups. (B) A heatmap of pathway enrichment signature in ABHD5 high and low subgroups. (C) ABHD5 +/+ (WT) and ABHD5 −/− (KO) colon epithelial cells (CCD841CON) were cultured in EBSS for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (D) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells colonies were exposed to EBSS culture, and the colony survival was calculated at the indicated time points by crystal violet and trypan blue exclusion assay. (E) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells were cultured in EBSS for 0, 3, 6 or 12 h , and the cell viability was determined by MTT assay. (F) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells stably transfected with a wild-type ABHD5 expression plasmid or control empty vector were cultured in EBSS for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (G) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (H) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells were cultured in EBSS for 3 h and analyzed by transmission electron microscopy. Arrows, autophagosomes; M, mitochondria; LD, lipid droplet; N, nucleus. The number of autophagosomes per cross-sectioned cell was counted. (I) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells were infected with GFP-RFP-LC3 adenovirus; 24 h after infection, high-content screen images showing RFP- and GFP-labeled LC3 staining in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells at different time points (2, 4, 8 and 16 h in the presence of EBSS culture). Scale bar: 5 μm. Statistical analysis showing autophagosomes, autolysosomes and the corresponding cell viability (J) in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells at the indicated timepoints. (K) Western blots of autophagy-related proteins (LC3-I, LC3-II, SQSTM1) in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells at 24 h following exposure to PBS, EBSS or EBSS+rapamycin (100 nM). (L) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells were cultured in EBSS in the presence of rapamycin (Rap, 100nM), dihydro-N-acetyl-d-erythro-sphingosine (NADS, 10 mM), brefeldin A (Bre A, 5 µM) or control PBS for 0, 3, 6, 12 or 18 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. Unless noted, all bar plots in the figure are mean ± SEM of n biological replicates. (*, p
    Figure Legend Snippet: Loss of ABHD5 suppresses CASP-independent cell death induced by nutrition deprivation. (A) GSEA plot of autophagy, apoptosis and WNT signaling pathways between ABHD5 high and ABHD5 low subgroups. (B) A heatmap of pathway enrichment signature in ABHD5 high and low subgroups. (C) ABHD5 +/+ (WT) and ABHD5 −/− (KO) colon epithelial cells (CCD841CON) were cultured in EBSS for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (D) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells colonies were exposed to EBSS culture, and the colony survival was calculated at the indicated time points by crystal violet and trypan blue exclusion assay. (E) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells were cultured in EBSS for 0, 3, 6 or 12 h , and the cell viability was determined by MTT assay. (F) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells stably transfected with a wild-type ABHD5 expression plasmid or control empty vector were cultured in EBSS for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (G) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (H) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells were cultured in EBSS for 3 h and analyzed by transmission electron microscopy. Arrows, autophagosomes; M, mitochondria; LD, lipid droplet; N, nucleus. The number of autophagosomes per cross-sectioned cell was counted. (I) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells were infected with GFP-RFP-LC3 adenovirus; 24 h after infection, high-content screen images showing RFP- and GFP-labeled LC3 staining in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells at different time points (2, 4, 8 and 16 h in the presence of EBSS culture). Scale bar: 5 μm. Statistical analysis showing autophagosomes, autolysosomes and the corresponding cell viability (J) in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells at the indicated timepoints. (K) Western blots of autophagy-related proteins (LC3-I, LC3-II, SQSTM1) in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells at 24 h following exposure to PBS, EBSS or EBSS+rapamycin (100 nM). (L) ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells were cultured in EBSS in the presence of rapamycin (Rap, 100nM), dihydro-N-acetyl-d-erythro-sphingosine (NADS, 10 mM), brefeldin A (Bre A, 5 µM) or control PBS for 0, 3, 6, 12 or 18 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. Unless noted, all bar plots in the figure are mean ± SEM of n biological replicates. (*, p

    Techniques Used: Cell Culture, Trypan Blue Exclusion Assay, MTT Assay, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Transmission Assay, Electron Microscopy, Infection, Labeling, Staining, Western Blot

    Loss of ABHD5 promotes DNA damage and genome instability in an autophagy-dependent manner. (A) ABHD5 +/+ (WT) and ABHD5 −/− (KO) colon epithelial cells (CCD841CON) were cultured in EBSS for 12 h, and the DNA damage was measured by alkaline single cell gel electrophoresis (Comet) assay. Error bars of statistical analysis of tail length represent standard error. (B) Immunostaining of γH2AFX (green) in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells at the indicated time points after exposure to EBSS. DAPI was used to stain the nuclei. (C) Quantification of γH2AFX-positive foci from (B). (D) Western blots of DNA damage-associated proteins in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells 24 h following exposure to EBSS. (E) Representative photomicrographs of ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells stained by indirect immunofluorescence for microtubules (anti-TUBULIN), DNA (DAPI), and centrosome-related protein (anti-NDEL1/NUDEL). Note the extensive microtubule network, the heterogeneous nuclear size, and the abnormalities in NDEL1 expression in ABHD5 −/− (KO) cells. (F) Quantification of centrosome numbers in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells. Percentage of cells with normal centrosome numbers (one or 2; blue bar) and with supernumerary centrosomes (more than 2; red bar). (G) Flow cytometry analysis of a panel of ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells showing their DNA content. (H) Analysis of total DNA content and aneuploid DNA content in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells. (I) Statistical analysis of tail length, γH2AFX-positive foci and centrosome numbers in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells matched with or without rapamycin treatment (50 nmol/L, 24 h). Rapamycin treatment significantly reversed the tail length, γH2AFX-positive foci and centrosome numbers in ABHD5 −/− (KO) cells to the level of control cells. (*, p
    Figure Legend Snippet: Loss of ABHD5 promotes DNA damage and genome instability in an autophagy-dependent manner. (A) ABHD5 +/+ (WT) and ABHD5 −/− (KO) colon epithelial cells (CCD841CON) were cultured in EBSS for 12 h, and the DNA damage was measured by alkaline single cell gel electrophoresis (Comet) assay. Error bars of statistical analysis of tail length represent standard error. (B) Immunostaining of γH2AFX (green) in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells at the indicated time points after exposure to EBSS. DAPI was used to stain the nuclei. (C) Quantification of γH2AFX-positive foci from (B). (D) Western blots of DNA damage-associated proteins in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells 24 h following exposure to EBSS. (E) Representative photomicrographs of ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells stained by indirect immunofluorescence for microtubules (anti-TUBULIN), DNA (DAPI), and centrosome-related protein (anti-NDEL1/NUDEL). Note the extensive microtubule network, the heterogeneous nuclear size, and the abnormalities in NDEL1 expression in ABHD5 −/− (KO) cells. (F) Quantification of centrosome numbers in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells. Percentage of cells with normal centrosome numbers (one or 2; blue bar) and with supernumerary centrosomes (more than 2; red bar). (G) Flow cytometry analysis of a panel of ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells showing their DNA content. (H) Analysis of total DNA content and aneuploid DNA content in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells. (I) Statistical analysis of tail length, γH2AFX-positive foci and centrosome numbers in ABHD5 +/+ (WT) and ABHD5 −/− (KO) CCD841CON cells matched with or without rapamycin treatment (50 nmol/L, 24 h). Rapamycin treatment significantly reversed the tail length, γH2AFX-positive foci and centrosome numbers in ABHD5 −/− (KO) cells to the level of control cells. (*, p

    Techniques Used: Cell Culture, Alkaline Single Cell Gel Electrophoresis, Single Cell Gel Electrophoresis, Immunostaining, Staining, Western Blot, Immunofluorescence, Expressing, Flow Cytometry, Cytometry

    7) Product Images from "Inhibition of autophagy-attenuated calcium oxalate crystal-induced renal tubular epithelial cell injury in vivo and in vitro"

    Article Title: Inhibition of autophagy-attenuated calcium oxalate crystal-induced renal tubular epithelial cell injury in vivo and in vitro

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23383

    Effects of 3-methyladenine and rapamycin on CaOx crystal-induced HK-2 cell injury HK-2 cells were incubated with CaOx crystals (4 mM) for 24 h in the absence or presence of 3-methyladenine (3-MA, 5 mM) or rapamycin (Rapa, 10 μM). ( A ) DAPI staining was used to examine cell and nuclear morphology to analyze apoptosis. White arrows indicated apoptosis of HK-2 cells; scale bar: 50 μm. ( B ) Quantitative analysis of CaOx crystals induced apoptosis. ( C ) Cell viability was measured by CCK-8 assay. ( D ) The levels of LDH in culture supernatant were determined using the LDH assay. Data are presented as the mean ± SD from three experiments. *** P
    Figure Legend Snippet: Effects of 3-methyladenine and rapamycin on CaOx crystal-induced HK-2 cell injury HK-2 cells were incubated with CaOx crystals (4 mM) for 24 h in the absence or presence of 3-methyladenine (3-MA, 5 mM) or rapamycin (Rapa, 10 μM). ( A ) DAPI staining was used to examine cell and nuclear morphology to analyze apoptosis. White arrows indicated apoptosis of HK-2 cells; scale bar: 50 μm. ( B ) Quantitative analysis of CaOx crystals induced apoptosis. ( C ) Cell viability was measured by CCK-8 assay. ( D ) The levels of LDH in culture supernatant were determined using the LDH assay. Data are presented as the mean ± SD from three experiments. *** P

    Techniques Used: Incubation, Staining, CCK-8 Assay, Lactate Dehydrogenase Assay

    Effects of 3-methyladenine and rapamycin on CaOx crystal-induced autophagy ( A ) HK-2 cells were transduced with Ad-mRFP-GFP-LC3 and then treated with vehicle or CaOx crystals (4 mM), with or without 3-methyladenine (3-MA, 5 mM) or rapamycin (Rapa, 10 μM) for 24 h. Under confocal microscopy, GFP dots displayed as green fluorescence while mRFP dots are red. In the merged images, autophagosomes and autolysosomes are labeled with yellow and red dots, respectively; scale bar: 20 μm. ( B ) Mean numbers of GFP and mRFP dots per cell. ( C ) Mean numbers of autophagosomes and autolysosomes per cell. ( D and E ) Representative immunoblot and quantification analysis of LC3-II as assayed after exposure to vehicle or CaOx crystals (4 mM) in the absence or presence of 3-methyladenine (3-MA, 5 mM) or rapamycin (Rapa, 10 μM) for 24 h. Data are presented as the mean ± SD from three experiments. ** P
    Figure Legend Snippet: Effects of 3-methyladenine and rapamycin on CaOx crystal-induced autophagy ( A ) HK-2 cells were transduced with Ad-mRFP-GFP-LC3 and then treated with vehicle or CaOx crystals (4 mM), with or without 3-methyladenine (3-MA, 5 mM) or rapamycin (Rapa, 10 μM) for 24 h. Under confocal microscopy, GFP dots displayed as green fluorescence while mRFP dots are red. In the merged images, autophagosomes and autolysosomes are labeled with yellow and red dots, respectively; scale bar: 20 μm. ( B ) Mean numbers of GFP and mRFP dots per cell. ( C ) Mean numbers of autophagosomes and autolysosomes per cell. ( D and E ) Representative immunoblot and quantification analysis of LC3-II as assayed after exposure to vehicle or CaOx crystals (4 mM) in the absence or presence of 3-methyladenine (3-MA, 5 mM) or rapamycin (Rapa, 10 μM) for 24 h. Data are presented as the mean ± SD from three experiments. ** P

    Techniques Used: Transduction, Confocal Microscopy, Fluorescence, Labeling

    8) Product Images from "Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes"

    Article Title: Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2011.162

    Mutant Htt-552 was reduced by enhanced autophagy. (A) Astrocytes were harvested after being infected for 72 h and treated with rapamycin (0.2 μg/mL) or 3-MA (10 mmol/L) for the last 24 h. Western blotting results showed increased LC3II/LC3I with treatment of rapamycin and decreased LC3II/LC3I with the treatment of 3-MA ( c P
    Figure Legend Snippet: Mutant Htt-552 was reduced by enhanced autophagy. (A) Astrocytes were harvested after being infected for 72 h and treated with rapamycin (0.2 μg/mL) or 3-MA (10 mmol/L) for the last 24 h. Western blotting results showed increased LC3II/LC3I with treatment of rapamycin and decreased LC3II/LC3I with the treatment of 3-MA ( c P

    Techniques Used: Mutagenesis, Infection, Western Blot

    Recovered expression of GLT-1 and glutamate uptake by autophagy stimulator. The densities of respective protein bands in each group were analyzed with Sigma Scan Pro 5, and β-actin was used as a reference. All the results are shown as mean±SD ( n =3). (A) Astrocytes were harvested after being infected for 72 h and treated with rapamycin (0.2 μg/mL) during the last 24 h. (A) Western blotting showing recovery of GLT-1 expression with stimulation of autophagy ( c P
    Figure Legend Snippet: Recovered expression of GLT-1 and glutamate uptake by autophagy stimulator. The densities of respective protein bands in each group were analyzed with Sigma Scan Pro 5, and β-actin was used as a reference. All the results are shown as mean±SD ( n =3). (A) Astrocytes were harvested after being infected for 72 h and treated with rapamycin (0.2 μg/mL) during the last 24 h. (A) Western blotting showing recovery of GLT-1 expression with stimulation of autophagy ( c P

    Techniques Used: Expressing, Infection, Western Blot

    9) Product Images from "Rapamycin induces differentiation of glioma stem/progenitor cells by activating autophagy"

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

    Journal: Chinese Journal of Cancer

    doi: 10.5732/cjc.011.10234

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

    Techniques Used:

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

    Techniques Used: In Vivo, Mouse Assay, Transmission Electron Microscopy

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

    Techniques Used: Microscopy, Expressing

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

    Techniques Used: In Vivo, Immunohistochemistry, Staining, Expressing

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

    Techniques Used: Quantitative RT-PCR, Immunocytochemistry

    10) Product Images from "Characterization of stress response in human retinal epithelial cells"

    Article Title: Characterization of stress response in human retinal epithelial cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2012.01652.x

    HMA induces autophagy. ( A ) ARPE 19 cells were untreated or treated with rapamycin for 2 hrs, with HMA or etoposide for 24 hrs and then probed with anti-LC3 antibody. Scale bar 25 μm. ( B ) ARPE-19 cells were treated as before and analysed by Western blot using anti-beclin 1, ATG-7, AGT5-12 and LC3 antibodies. γ tubulin was used as a loading control (left panel). On middle and right panels, cells were treated as before and analysed using anti-ERK and phospho-ERK (*) antibodies (middle panel) or with anti JNK1 or phosphorylated JNK1 (*) antibodies (right panel). Under the western images the quantification of the bands is reported showing a significant increase of Beclin 1 (Means are different from each other as calculated from a one-way anova test ( P
    Figure Legend Snippet: HMA induces autophagy. ( A ) ARPE 19 cells were untreated or treated with rapamycin for 2 hrs, with HMA or etoposide for 24 hrs and then probed with anti-LC3 antibody. Scale bar 25 μm. ( B ) ARPE-19 cells were treated as before and analysed by Western blot using anti-beclin 1, ATG-7, AGT5-12 and LC3 antibodies. γ tubulin was used as a loading control (left panel). On middle and right panels, cells were treated as before and analysed using anti-ERK and phospho-ERK (*) antibodies (middle panel) or with anti JNK1 or phosphorylated JNK1 (*) antibodies (right panel). Under the western images the quantification of the bands is reported showing a significant increase of Beclin 1 (Means are different from each other as calculated from a one-way anova test ( P

    Techniques Used: Western Blot

    Role of autophagy in HMA response. ( A ) ARPE-19 cells were treated with HMA in the absence or presence of 1 μM rapamycin or 5 mM 3-MA. After 2 or 4 hrs of treatment, cells were trypsinized and counted. A total of 1000 cells were seeded into 6-well plates. Seven days after seeding, cells were stained with cresyl violet. Controls using rapamycin or 3-MA alone have show no difference with the control (not shown) ( B ) Using image analysis, the surface covered by the cells was measured and plotted. For 2 hrs of treatment all means were significantly different ( P
    Figure Legend Snippet: Role of autophagy in HMA response. ( A ) ARPE-19 cells were treated with HMA in the absence or presence of 1 μM rapamycin or 5 mM 3-MA. After 2 or 4 hrs of treatment, cells were trypsinized and counted. A total of 1000 cells were seeded into 6-well plates. Seven days after seeding, cells were stained with cresyl violet. Controls using rapamycin or 3-MA alone have show no difference with the control (not shown) ( B ) Using image analysis, the surface covered by the cells was measured and plotted. For 2 hrs of treatment all means were significantly different ( P

    Techniques Used: Staining

    11) Product Images from "Data of intracellular insulin protein reduced by autophagy in INS-1E cells"

    Article Title: Data of intracellular insulin protein reduced by autophagy in INS-1E cells

    Journal: Data in Brief

    doi: 10.1016/j.dib.2016.07.008

    Expression of LC3 and insulin in the presence or absence of rapamycin, in rat INS-1E insulinoma cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with or without rapamycin (20–80 nM) for 24 h at 37 °C with 5% CO 2 . LC3 and insulin were measured by Western blot (A). The relative amounts of LC3 (B) and insulin (C) were quantified as described in the Methods section. Data represent mean±SD of three experiments. * p
    Figure Legend Snippet: Expression of LC3 and insulin in the presence or absence of rapamycin, in rat INS-1E insulinoma cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with or without rapamycin (20–80 nM) for 24 h at 37 °C with 5% CO 2 . LC3 and insulin were measured by Western blot (A). The relative amounts of LC3 (B) and insulin (C) were quantified as described in the Methods section. Data represent mean±SD of three experiments. * p

    Techniques Used: Expressing, Incubation, Western Blot

    Expression of cellular insulin following bafilomycin A1 or 3-methyladenine treatment with rapamycin in rat INS-1E insulinoma cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with or without rapamycin (80 nM) for 24 h, and with or without bafilomycin A1 (100 nM) or 3-methyladenine (10 mM) for 6 h at 37 °C with 5% CO 2 . Insulin was measured by Western blot (A) and the relative amounts of insulin (B) were quantified as described in the Methods section. Data represent mean ± SD of three experiments. * p
    Figure Legend Snippet: Expression of cellular insulin following bafilomycin A1 or 3-methyladenine treatment with rapamycin in rat INS-1E insulinoma cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with or without rapamycin (80 nM) for 24 h, and with or without bafilomycin A1 (100 nM) or 3-methyladenine (10 mM) for 6 h at 37 °C with 5% CO 2 . Insulin was measured by Western blot (A) and the relative amounts of insulin (B) were quantified as described in the Methods section. Data represent mean ± SD of three experiments. * p

    Techniques Used: Expressing, Incubation, Western Blot

    12) Product Images from "Comparison of rapamycin schedules in mice on high-fat diet"

    Article Title: Comparison of rapamycin schedules in mice on high-fat diet

    Journal: Cell Cycle

    doi: 10.4161/15384101.2014.970491

    Metabolic profiles of 16 month-old mice on high fat diet 8 months from the beginning of the of treatment with different schedules of rapamycin or metformin. p values: the differences with control group (HFD). Glucose, insulin and triglycerides were determined
    Figure Legend Snippet: Metabolic profiles of 16 month-old mice on high fat diet 8 months from the beginning of the of treatment with different schedules of rapamycin or metformin. p values: the differences with control group (HFD). Glucose, insulin and triglycerides were determined

    Techniques Used: Mouse Assay

    Body weight and leptin levels of female mice treated with different schedules of rapamycin or metformin. Weight (grams) of 16-month old mice, 8 months after beginning of the treatments. Low (5%) fat (LF) diet (LF); all other groups received high (60%)
    Figure Legend Snippet: Body weight and leptin levels of female mice treated with different schedules of rapamycin or metformin. Weight (grams) of 16-month old mice, 8 months after beginning of the treatments. Low (5%) fat (LF) diet (LF); all other groups received high (60%)

    Techniques Used: Mouse Assay

    Blood IGF1 and triglyceride levels in 23 month-old mice on high fat diet 15 months from the beginning of treatment with different schedules of rapamycin or metformin. IGF1 and triglyceride concentration. Data are mean ± SE.
    Figure Legend Snippet: Blood IGF1 and triglyceride levels in 23 month-old mice on high fat diet 15 months from the beginning of treatment with different schedules of rapamycin or metformin. IGF1 and triglyceride concentration. Data are mean ± SE.

    Techniques Used: Mouse Assay, Concentration Assay

    Blood glucose and insulin levels in 23 month-old mice on high fat diet 15 months from the beginning of the treatment with different schedules of rapamycin or metformin. Insulin ( A and B ) and glucose ( C and D ) concentrations were determined in fasting
    Figure Legend Snippet: Blood glucose and insulin levels in 23 month-old mice on high fat diet 15 months from the beginning of the treatment with different schedules of rapamycin or metformin. Insulin ( A and B ) and glucose ( C and D ) concentrations were determined in fasting

    Techniques Used: Mouse Assay

    Cardiac levels of p-S6 in mice on HFD. Immunoblot analysis of protein lysates from the heart of 23 month-old female mice on high fat diet: control – untreated; R/ ip – treated with rapamycin i.p 3 times a week every other week;
    Figure Legend Snippet: Cardiac levels of p-S6 in mice on HFD. Immunoblot analysis of protein lysates from the heart of 23 month-old female mice on high fat diet: control – untreated; R/ ip – treated with rapamycin i.p 3 times a week every other week;

    Techniques Used: Mouse Assay

    13) Product Images from "Small Molecules Affect Human Dental Pulp Stem Cell Properties Via Multiple Signaling Pathways"

    Article Title: Small Molecules Affect Human Dental Pulp Stem Cell Properties Via Multiple Signaling Pathways

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2012.0426

    Western blot analysis of signaling events in DPSCs after treatment with SC1 or rapamycin (Rapa). Cells were treated with 300 nM SC1 (A) or 900 nM Rapa (B) for 5 days and subjected to protein extraction for detection of signaling factors
    Figure Legend Snippet: Western blot analysis of signaling events in DPSCs after treatment with SC1 or rapamycin (Rapa). Cells were treated with 300 nM SC1 (A) or 900 nM Rapa (B) for 5 days and subjected to protein extraction for detection of signaling factors

    Techniques Used: Western Blot, Protein Extraction

    Cell viability analysis after treatment with small molecules. Dental pulp stem cells (DPSCs) were seeded in 12-well plates and treated with BIO, Rapa (rapamycin), or SC1 of various concentrations. At different time points, cells were harvested and the
    Figure Legend Snippet: Cell viability analysis after treatment with small molecules. Dental pulp stem cells (DPSCs) were seeded in 12-well plates and treated with BIO, Rapa (rapamycin), or SC1 of various concentrations. At different time points, cells were harvested and the

    Techniques Used:

    14) Product Images from "Pleiotropic effects of miR-183∼96∼182 converge to regulate cell survival, proliferation and migration in medulloblastoma"

    Article Title: Pleiotropic effects of miR-183∼96∼182 converge to regulate cell survival, proliferation and migration in medulloblastoma

    Journal: Acta neuropathologica

    doi: 10.1007/s00401-012-0969-5

    miR-183∼96∼182 cluster regulates the PI3K/AKT/mTOR signaling axis. a Immunofluorescence staining shows elevated AKT1 (red) and decreased AKT2 (green) upon knockdown of the microRNA cluster in D458 medulloblastoma cells. b Western Blot showing increased AKT1 and decreased AKT2 in pooled miR-183∼96∼182 knockdown compared to the scrambled control. c Increased migration upon forced expression of miR-183∼96∼182 cluster in DAOY cells is negated by rapamycin. d Impeded migration upon rapamycin treatment in UW426 cells comparable to the effects of miR-183∼96∼182 knockdown. Bar graphs represent migration into wounds measured in an unbiased manner based on pixel intensity. P-values (Fisher's exact test) indicate significance relative to scramble control, **p-value
    Figure Legend Snippet: miR-183∼96∼182 cluster regulates the PI3K/AKT/mTOR signaling axis. a Immunofluorescence staining shows elevated AKT1 (red) and decreased AKT2 (green) upon knockdown of the microRNA cluster in D458 medulloblastoma cells. b Western Blot showing increased AKT1 and decreased AKT2 in pooled miR-183∼96∼182 knockdown compared to the scrambled control. c Increased migration upon forced expression of miR-183∼96∼182 cluster in DAOY cells is negated by rapamycin. d Impeded migration upon rapamycin treatment in UW426 cells comparable to the effects of miR-183∼96∼182 knockdown. Bar graphs represent migration into wounds measured in an unbiased manner based on pixel intensity. P-values (Fisher's exact test) indicate significance relative to scramble control, **p-value

    Techniques Used: Immunofluorescence, Staining, Western Blot, Migration, Expressing

    15) Product Images from "Oligomer procyanidins (F2) repress HIF-1α expression in human U87 glioma cells by inhibiting the EGFR/ AKT/mTOR and MAPK/ERK1/2 signaling pathways in vitro and in vivo"

    Article Title: Oligomer procyanidins (F2) repress HIF-1α expression in human U87 glioma cells by inhibiting the EGFR/ AKT/mTOR and MAPK/ERK1/2 signaling pathways in vitro and in vivo

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19654

    The effects of F2 on the EGFR/PI3K/AKT/mTOR and MAPK pathways (A, B) Effects of F2 on hypoxia-induced signaling pathways related to the expression of HIF-1α in U87 cells. Cells were pretreated with the indicated concentrations of F2 for 1 h, then incubated under normoxic and hypoxic conditions for 1 h. Total proteins were then collected for western blotting using antibodies as indicated. (C) Effects of inhibitors on hypoxia-induced expression of HIF-1α in U87 cells. Cells were pretreated with LY (LY294002, 100 μM), Rap (Rapamycin, 100 nM), PD (PD98059, 100 μM) and AG1478 for 1 h, and then incubated under normoxic or hypoxic conditions for 16 h. Total proteins were subjected to western blotting using antibodies against HIF-1α. (D) Cells were pretreated with or without LY (LY294002, 100 μM) or PD (PD98059, 100 μM), then co-treated with or without F2 (10 μg/ml) for 16 h under hypoxic conditions. LY’ and PD’ indicate cells treated with inhibitors and F2. Total proteins were collected for western blot analysis.
    Figure Legend Snippet: The effects of F2 on the EGFR/PI3K/AKT/mTOR and MAPK pathways (A, B) Effects of F2 on hypoxia-induced signaling pathways related to the expression of HIF-1α in U87 cells. Cells were pretreated with the indicated concentrations of F2 for 1 h, then incubated under normoxic and hypoxic conditions for 1 h. Total proteins were then collected for western blotting using antibodies as indicated. (C) Effects of inhibitors on hypoxia-induced expression of HIF-1α in U87 cells. Cells were pretreated with LY (LY294002, 100 μM), Rap (Rapamycin, 100 nM), PD (PD98059, 100 μM) and AG1478 for 1 h, and then incubated under normoxic or hypoxic conditions for 16 h. Total proteins were subjected to western blotting using antibodies against HIF-1α. (D) Cells were pretreated with or without LY (LY294002, 100 μM) or PD (PD98059, 100 μM), then co-treated with or without F2 (10 μg/ml) for 16 h under hypoxic conditions. LY’ and PD’ indicate cells treated with inhibitors and F2. Total proteins were collected for western blot analysis.

    Techniques Used: Expressing, Incubation, Western Blot

    The effects of F2 on EGF- and FBS-induced HIF-1α expression (A, B) Effects of F2 on EGF- or FBS-induced signaling related to the expression of HIF-1α in U87 cells. Cells starved for 24 h were pretreated with the indicated concentrations of F2 for 1 h, followed by incubation with EGF or FBS for16 h or 1 h respectively. HIF-1α and the phosphorylated and total levels of EGFR, ERK, and AKT were determined by western blotting. (C) Effects of kinase inhibitors on EGF- or FBS-induced expression of HIF-1α in U87 cells. Cells starved for 24 h were pretreated with LY (LY294002, 100 μM), Rap (Rapamycin, 100 nM), PD (PD98059, 100 μM) and AG1478(5-50 μM) for 1 h, then incubated with EGF or FBS for16 h. Total protein extracts were subjected to western blotting using antibodies against HIF-1α or β-actin. (D) U87 cells were pre-treated with the indicated concentrations of AG1478 for 1 h, then incubated with EGF or FBS for 16 h. Total protein extracts were analyzed by western blotting with the indicated antibodies.
    Figure Legend Snippet: The effects of F2 on EGF- and FBS-induced HIF-1α expression (A, B) Effects of F2 on EGF- or FBS-induced signaling related to the expression of HIF-1α in U87 cells. Cells starved for 24 h were pretreated with the indicated concentrations of F2 for 1 h, followed by incubation with EGF or FBS for16 h or 1 h respectively. HIF-1α and the phosphorylated and total levels of EGFR, ERK, and AKT were determined by western blotting. (C) Effects of kinase inhibitors on EGF- or FBS-induced expression of HIF-1α in U87 cells. Cells starved for 24 h were pretreated with LY (LY294002, 100 μM), Rap (Rapamycin, 100 nM), PD (PD98059, 100 μM) and AG1478(5-50 μM) for 1 h, then incubated with EGF or FBS for16 h. Total protein extracts were subjected to western blotting using antibodies against HIF-1α or β-actin. (D) U87 cells were pre-treated with the indicated concentrations of AG1478 for 1 h, then incubated with EGF or FBS for 16 h. Total protein extracts were analyzed by western blotting with the indicated antibodies.

    Techniques Used: Expressing, Incubation, Western Blot

    16) Product Images from "Bortezomib initiates endoplasmic reticulum stress, elicits autophagy and death in Echinococcus granulosus larval stage"

    Article Title: Bortezomib initiates endoplasmic reticulum stress, elicits autophagy and death in Echinococcus granulosus larval stage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181528

    Effect of combinatorial treatment of bortezomib (Bz) and rapamycin (Rm) on Echinococcus cell viability. (A) Effect of Rm and Bz on E . granulosus protoscolex viability at 10 μM for individual drugs and 5 μM or 10 μM of each drug for a combined protocol over 7 days (the drugs were replenished after 3 days). Protoscoleces incubated in culture medium containing 1:1.000 DMSO were used as controls. Each point represents the mean percentage of vital protoscoleces from three different experiments. Asterisks indicate a statistically significant difference (P
    Figure Legend Snippet: Effect of combinatorial treatment of bortezomib (Bz) and rapamycin (Rm) on Echinococcus cell viability. (A) Effect of Rm and Bz on E . granulosus protoscolex viability at 10 μM for individual drugs and 5 μM or 10 μM of each drug for a combined protocol over 7 days (the drugs were replenished after 3 days). Protoscoleces incubated in culture medium containing 1:1.000 DMSO were used as controls. Each point represents the mean percentage of vital protoscoleces from three different experiments. Asterisks indicate a statistically significant difference (P

    Techniques Used: Incubation

    17) Product Images from "Peripheral Administration of Translation Inhibitors Reverses Increased Hyperalgesia in a Model of Chronic Pain in the Rat"

    Article Title: Peripheral Administration of Translation Inhibitors Reverses Increased Hyperalgesia in a Model of Chronic Pain in the Rat

    Journal: The journal of pain : official journal of the American Pain Society

    doi: 10.1016/j.jpain.2013.01.779

    Reversal of carrageenan-induced hyperalgesic priming by local injection of the mTOR inhibitor rapamycin
    Figure Legend Snippet: Reversal of carrageenan-induced hyperalgesic priming by local injection of the mTOR inhibitor rapamycin

    Techniques Used: Injection

    Prevention of carrageenan-induced hyperalgesic priming by local injection of the mTOR inhibitor rapamycin
    Figure Legend Snippet: Prevention of carrageenan-induced hyperalgesic priming by local injection of the mTOR inhibitor rapamycin

    Techniques Used: Injection

    18) Product Images from "Pentraxin-3 Is a TSH-Inducible Protein in Human Fibrocytes and Orbital Fibroblasts"

    Article Title: Pentraxin-3 Is a TSH-Inducible Protein in Human Fibrocytes and Orbital Fibroblasts

    Journal: Endocrinology

    doi: 10.1210/en.2015-1399

    A, Fibrocytes were pretreated with nothing or AKTi (1 μM), LY294002 (10 μM), AG490 (75 μM), rapamycin (RAPA) (20 nM) for 1 hour before incubation with bTSH (5 mIU/mL) for 6 hours. RNA was subjected to RT-PCR for PTX3 mRNA. Data
    Figure Legend Snippet: A, Fibrocytes were pretreated with nothing or AKTi (1 μM), LY294002 (10 μM), AG490 (75 μM), rapamycin (RAPA) (20 nM) for 1 hour before incubation with bTSH (5 mIU/mL) for 6 hours. RNA was subjected to RT-PCR for PTX3 mRNA. Data

    Techniques Used: Incubation, Reverse Transcription Polymerase Chain Reaction

    19) Product Images from "A Complex between Atg7 and Caspase-9"

    Article Title: A Complex between Atg7 and Caspase-9

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.536854

    The expression level of C9 impacts the pattern and expression level of LC3 under basal conditions or autophagy induced by rapamycin or starvation. A and B , reduced expression of LC3 vesicles in starved or rapamycin-treated C9 KO MEF (2–4 μ
    Figure Legend Snippet: The expression level of C9 impacts the pattern and expression level of LC3 under basal conditions or autophagy induced by rapamycin or starvation. A and B , reduced expression of LC3 vesicles in starved or rapamycin-treated C9 KO MEF (2–4 μ

    Techniques Used: Expressing

    20) Product Images from "Autophagic degradation of farnesylated prelamin A as a therapeutic approach to lamin-linked progeria"

    Article Title: Autophagic degradation of farnesylated prelamin A as a therapeutic approach to lamin-linked progeria

    Journal: European Journal of Histochemistry : EJH

    doi: 10.4081/ejh.2011.e36

    Nuclear envelope/lamina proteins in rapamycin treated cells. A) Western Blotting analysis of LAP2α (LAP2 ), lamin B1 (lamin B1), lamin B2 (lamin B2), emerin (emerin) and Barrier-to-autointegration factor (BAF) in control (control) and HGPS cells (HGPS) untreated (−) or treated (+) with rapamycin. Actin was detected as protein loading control. Immunolabeled bands are shown; B) densitometric analysis of LAP2α immunolabeled bands detected in Western blotting analysis performed in control and HGPS untreated (−) or rapamycin treated cells (+). Asterisk indicates statistically significant difference, P=0.0319 for HGPS + rapamycin (+) vs untreated HGPS (−); C) prelamin A and LAP2α immunofluorescence labeling performed in untreated (−) or rapamycin treated (+) control cells. Prelamin A was evaluated using a goat-polyclonal antibody visualized by TRITC-conjugated secondary antibody (red). LAP2α distribution was evaluated using a rabbitpolyclonal antibody visualized by FITC-conjugated secondary antibody (green); D) progerin and LAP2α immunolabeling detection performed in untreated (−) or rapamycin treated (+) HGPS cells. Progerin (progerin) detection was performed using a mouse-monoclonal antibody visualized by Cy3-conjugated secondary antibody (red). LAP2α distribution was evaluated using a rabbit-polyclonal antibody visualized by FITC-conjugated secondary antibody (green). In untreated HGPS cells progerin staining was observed, while LAP2α labeling was decreased. Rapamycin treatment dramatically reduced progerin labeling and restored LAP2α staining levels; E) Lamin A/C and farnesylated-prelamin A detection performed in untreated (−) or rapamycin treated (+) control cells. Lamin A/C (lamin A/C) was evaluated using a goat-polyclonal antibody visualized by TRITC-conjugated secondary antibody (red). Farnesylated-prelamin A (F-prelamin A) staining was performed using a rabbit-polyclonal antibody visualized by FITC-conjugated secondary antibody (green). Lamin A/C was detected in untreated or treated cells at the same levels. F-prelamin A was undetected in both samples; F) Lamin A/C and farnesylated-prelamin A detection performed in untreated (−) or rapamycin treated (+) HGPS cells. Lamin A/C (lamin A/C) was evaluated using a goat-polyclonal antibody visualized by TRITC-conjugated secondary antibody (red). Farnesylated-prelamin A (F-prelamin A) staining was performed using a rabbit-polyclonal antibody visualized by FITC-conjugated secondary antibody (green). Lamin A/C staining was observed at the nuclear lamina of both untreated and treated cells. F-prelamin A staining observed in untreated cells was strongly reduced by rapamycin treatment; G) prelamin A BAF detection performed in untreated (−) or rapamycin treated (+) control cells. Prelamin A was stained by a goat-polyclonal antibody visualized by TRITC-conjugated secondary antibody (red). BAF immunolabeling detection was performed by a rabbit-polyclonal antibody visualized by FITC-conjugated secondary antibody (green). Prelamin A was undetectable in untreated or rapamycin treated cells. BAF showed a normal cellular distribution in both samples; H) prelamin A and Barrier-to-autointegration (BAF) detection was performed in untreated (−) or rapamycin treated (+) HGPS cells. Prelamin A was stained by a goat-polyclonal antibody visualized by TRITC-conjugated secondary antibody (red). BAF was labeled by rabbit-polyclonal antibody and visualized by FITC-conjugated secondary antibody (green). Prelamin A staining was present at the nuclear lamina of untreated cells while in rapamycin treated cells lamin A precursor was undetectable. BAF nuclear localization was observed in HGPS untreated cells. The normal BAF nucleo-cytoplasmic cellular distribution was recovered by rapamycin treatment. In panel C, D, E, F,G and H nuclei were counterstained with DAPI. Scale bar, 10 µm.
    Figure Legend Snippet: Nuclear envelope/lamina proteins in rapamycin treated cells. A) Western Blotting analysis of LAP2α (LAP2 ), lamin B1 (lamin B1), lamin B2 (lamin B2), emerin (emerin) and Barrier-to-autointegration factor (BAF) in control (control) and HGPS cells (HGPS) untreated (−) or treated (+) with rapamycin. Actin was detected as protein loading control. Immunolabeled bands are shown; B) densitometric analysis of LAP2α immunolabeled bands detected in Western blotting analysis performed in control and HGPS untreated (−) or rapamycin treated cells (+). Asterisk indicates statistically significant difference, P=0.0319 for HGPS + rapamycin (+) vs untreated HGPS (−); C) prelamin A and LAP2α immunofluorescence labeling performed in untreated (−) or rapamycin treated (+) control cells. Prelamin A was evaluated using a goat-polyclonal antibody visualized by TRITC-conjugated secondary antibody (red). LAP2α distribution was evaluated using a rabbitpolyclonal antibody visualized by FITC-conjugated secondary antibody (green); D) progerin and LAP2α immunolabeling detection performed in untreated (−) or rapamycin treated (+) HGPS cells. Progerin (progerin) detection was performed using a mouse-monoclonal antibody visualized by Cy3-conjugated secondary antibody (red). LAP2α distribution was evaluated using a rabbit-polyclonal antibody visualized by FITC-conjugated secondary antibody (green). In untreated HGPS cells progerin staining was observed, while LAP2α labeling was decreased. Rapamycin treatment dramatically reduced progerin labeling and restored LAP2α staining levels; E) Lamin A/C and farnesylated-prelamin A detection performed in untreated (−) or rapamycin treated (+) control cells. Lamin A/C (lamin A/C) was evaluated using a goat-polyclonal antibody visualized by TRITC-conjugated secondary antibody (red). Farnesylated-prelamin A (F-prelamin A) staining was performed using a rabbit-polyclonal antibody visualized by FITC-conjugated secondary antibody (green). Lamin A/C was detected in untreated or treated cells at the same levels. F-prelamin A was undetected in both samples; F) Lamin A/C and farnesylated-prelamin A detection performed in untreated (−) or rapamycin treated (+) HGPS cells. Lamin A/C (lamin A/C) was evaluated using a goat-polyclonal antibody visualized by TRITC-conjugated secondary antibody (red). Farnesylated-prelamin A (F-prelamin A) staining was performed using a rabbit-polyclonal antibody visualized by FITC-conjugated secondary antibody (green). Lamin A/C staining was observed at the nuclear lamina of both untreated and treated cells. F-prelamin A staining observed in untreated cells was strongly reduced by rapamycin treatment; G) prelamin A BAF detection performed in untreated (−) or rapamycin treated (+) control cells. Prelamin A was stained by a goat-polyclonal antibody visualized by TRITC-conjugated secondary antibody (red). BAF immunolabeling detection was performed by a rabbit-polyclonal antibody visualized by FITC-conjugated secondary antibody (green). Prelamin A was undetectable in untreated or rapamycin treated cells. BAF showed a normal cellular distribution in both samples; H) prelamin A and Barrier-to-autointegration (BAF) detection was performed in untreated (−) or rapamycin treated (+) HGPS cells. Prelamin A was stained by a goat-polyclonal antibody visualized by TRITC-conjugated secondary antibody (red). BAF was labeled by rabbit-polyclonal antibody and visualized by FITC-conjugated secondary antibody (green). Prelamin A staining was present at the nuclear lamina of untreated cells while in rapamycin treated cells lamin A precursor was undetectable. BAF nuclear localization was observed in HGPS untreated cells. The normal BAF nucleo-cytoplasmic cellular distribution was recovered by rapamycin treatment. In panel C, D, E, F,G and H nuclei were counterstained with DAPI. Scale bar, 10 µm.

    Techniques Used: Western Blot, Immunolabeling, Immunofluorescence, Labeling, Staining

    Rapamycin reduces progerin level in HGPS cells. A) Western blotting evaluation of lamin A/C and progerin in control (CONTROL) and Hutchinson Gilford progeria cells (HGPS). Whole lysates from control and HGPS cells untreated (−) or treated (+) with rapamycin (Rapamycin), MG132 or chloroquine-diphosphate (Cq) were subjected to lamin A/C, LC3-B2 and actin antibodies detection; B) densitometric analysis of lamin A and C immunolabeled bands detected in control fibroblasts; C) densitometric analysis of lamin A and C immunolabeled bands detected in HGPS fibroblasts; P=0.0358 for lamin A (rapa), P=0.0298 for lamin A (Mg); D) densitometric analysis of progerin immunoblotted bands; P=0.0390 for progerin (rapa); P=0.0458 for progerin (Mg); E) prelamin A, FLAG and LC3-B2 protein levels evaluation in HEK-293 cells expressing FLAG-tagged wild type prelamin A (LA-WT) or progerin (LA-Δ50). Immunolabeled bands observed in untreated (−) or rapamycin (Rapamycin) and chloroquine-diphosphate (Cq) treated (+) cells are shown; F) densitometric of FLAG immunoblotted bands; G) RT-PCR analysis of ZMPSTE24 and LMNA mRNA expression in untreated (Nt) and rapamycintreated HGPS cells (Rapa) and control (control); 2 −ΔΔCT values are reported relative to untreated control samples. P=0.0236 for LA-Δ50 (rapa); H) ratio between ZMPSTE24 and LMNA mRNA expression. Values are means of duplicate experiments ± S.D. In B, C, D and F densitometric analysis of triplicate experiments was performed, and the mean values ± S.D. are reported; asterisk indicates statistically significant difference with respect to lamin A or progerin densitometry in untreated samples; statistical significance was calculated by the Mann-Whitney test vs untreated HGPS samples, or cells expressing LMNA constructs.
    Figure Legend Snippet: Rapamycin reduces progerin level in HGPS cells. A) Western blotting evaluation of lamin A/C and progerin in control (CONTROL) and Hutchinson Gilford progeria cells (HGPS). Whole lysates from control and HGPS cells untreated (−) or treated (+) with rapamycin (Rapamycin), MG132 or chloroquine-diphosphate (Cq) were subjected to lamin A/C, LC3-B2 and actin antibodies detection; B) densitometric analysis of lamin A and C immunolabeled bands detected in control fibroblasts; C) densitometric analysis of lamin A and C immunolabeled bands detected in HGPS fibroblasts; P=0.0358 for lamin A (rapa), P=0.0298 for lamin A (Mg); D) densitometric analysis of progerin immunoblotted bands; P=0.0390 for progerin (rapa); P=0.0458 for progerin (Mg); E) prelamin A, FLAG and LC3-B2 protein levels evaluation in HEK-293 cells expressing FLAG-tagged wild type prelamin A (LA-WT) or progerin (LA-Δ50). Immunolabeled bands observed in untreated (−) or rapamycin (Rapamycin) and chloroquine-diphosphate (Cq) treated (+) cells are shown; F) densitometric of FLAG immunoblotted bands; G) RT-PCR analysis of ZMPSTE24 and LMNA mRNA expression in untreated (Nt) and rapamycintreated HGPS cells (Rapa) and control (control); 2 −ΔΔCT values are reported relative to untreated control samples. P=0.0236 for LA-Δ50 (rapa); H) ratio between ZMPSTE24 and LMNA mRNA expression. Values are means of duplicate experiments ± S.D. In B, C, D and F densitometric analysis of triplicate experiments was performed, and the mean values ± S.D. are reported; asterisk indicates statistically significant difference with respect to lamin A or progerin densitometry in untreated samples; statistical significance was calculated by the Mann-Whitney test vs untreated HGPS samples, or cells expressing LMNA constructs.

    Techniques Used: Western Blot, Immunolabeling, Expressing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY, Construct

    Rapamycin treatment rescues the heterochromatin marker trimethyl-H3K9 in HGPS cells. Immunofluorescence labeling of trimethyl-H3K9 (H3K9) in control and HGPS cells untreated (untreated) or treated (treated) with rapamycin using a rabbit-polyclonal antibody visualized by FITC-conjugated secondary antibody (green). In control untreated cells (A) a proper trimethyl-H3K9 staining was observed, while a clearly detectable decrease of immunolabeling was observed in untreated HGPS cells (C). Rapamycin treatment of control cells did not affect trimethyl-H3K9 staining (E), while rescued trimethyl-H3K9 distribution in HGPS cells (G). Nuclei in B, D, F and H were counterstained with DAPI. Scale bar = 10 µm; I) The mean fluorescence intensity of 300 nuclei per sample stained for trimethyl-H3K9 was measured by the NIS software and plotted for untreated (NT) or rapamycin-treated (treated) control and HGPS nuclei. Asterisk indicates statistically significant difference, P=0.0331 for HGPS + rapamycin vs untreated HGPS.
    Figure Legend Snippet: Rapamycin treatment rescues the heterochromatin marker trimethyl-H3K9 in HGPS cells. Immunofluorescence labeling of trimethyl-H3K9 (H3K9) in control and HGPS cells untreated (untreated) or treated (treated) with rapamycin using a rabbit-polyclonal antibody visualized by FITC-conjugated secondary antibody (green). In control untreated cells (A) a proper trimethyl-H3K9 staining was observed, while a clearly detectable decrease of immunolabeling was observed in untreated HGPS cells (C). Rapamycin treatment of control cells did not affect trimethyl-H3K9 staining (E), while rescued trimethyl-H3K9 distribution in HGPS cells (G). Nuclei in B, D, F and H were counterstained with DAPI. Scale bar = 10 µm; I) The mean fluorescence intensity of 300 nuclei per sample stained for trimethyl-H3K9 was measured by the NIS software and plotted for untreated (NT) or rapamycin-treated (treated) control and HGPS nuclei. Asterisk indicates statistically significant difference, P=0.0331 for HGPS + rapamycin vs untreated HGPS.

    Techniques Used: Marker, Immunofluorescence, Labeling, Staining, Immunolabeling, Fluorescence, Software

    21) Product Images from "Coronavirus membrane-associated papain-like proteases induce autophagy through interacting with Beclin1 to negatively regulate antiviral innate immunity"

    Article Title: Coronavirus membrane-associated papain-like proteases induce autophagy through interacting with Beclin1 to negatively regulate antiviral innate immunity

    Journal: Protein & Cell

    doi: 10.1007/s13238-014-0104-6

    Autophagosome induced by CoV NL63 PLP2-TM in various cell lines . (A) PLP2-TM-V5 and pcDNA3.1-eGFP-LC3B were co-transfected respectively into HEK2923T, HeLa and MCF-7 cells. The cells were fixed at 48 h post-transfection and were analyzed for eGFP-LC3B positive autophagosome accumulation (green) using a confocal microscope as described in Fig. 1 B. (B) Quantification of cells displaying eGFP-LC3B puncta from one representative experiment that shown in Fig. 2A as described in Fig. 1 C. (C–E) HEK293T, HeLa and MCF-7 cells were transfectd with PLP2-TM-V5 or empty vector as a negative control for 48 h. These cells were also treated by 400 nmol/L Rapamycin to serve as a positive control for induction of autophagy. The cells were then lysed for Western blotting analysis using a rabbit anti-LC3 antibody to detect the endogenous LC3 expression (top panel in each Fig. 2C–E). The whole cell lysate (WCL) was blotted using anti-V5 antibodies to evaluate expression of PLP2-TM (middle panel in each Fig. 2C–E), and β-Actin was detected from whole cell lysate (WCL) as a loading control (bottom panel in each Fig. 2C to 2E)
    Figure Legend Snippet: Autophagosome induced by CoV NL63 PLP2-TM in various cell lines . (A) PLP2-TM-V5 and pcDNA3.1-eGFP-LC3B were co-transfected respectively into HEK2923T, HeLa and MCF-7 cells. The cells were fixed at 48 h post-transfection and were analyzed for eGFP-LC3B positive autophagosome accumulation (green) using a confocal microscope as described in Fig. 1 B. (B) Quantification of cells displaying eGFP-LC3B puncta from one representative experiment that shown in Fig. 2A as described in Fig. 1 C. (C–E) HEK293T, HeLa and MCF-7 cells were transfectd with PLP2-TM-V5 or empty vector as a negative control for 48 h. These cells were also treated by 400 nmol/L Rapamycin to serve as a positive control for induction of autophagy. The cells were then lysed for Western blotting analysis using a rabbit anti-LC3 antibody to detect the endogenous LC3 expression (top panel in each Fig. 2C–E). The whole cell lysate (WCL) was blotted using anti-V5 antibodies to evaluate expression of PLP2-TM (middle panel in each Fig. 2C–E), and β-Actin was detected from whole cell lysate (WCL) as a loading control (bottom panel in each Fig. 2C to 2E)

    Techniques Used: Transfection, Microscopy, Plasmid Preparation, Negative Control, Positive Control, Western Blot, Expressing

    Induction of autophagy by various coronaviral PLPs that is independent on the protease activity . (A) The plasmids of HCoV-NL63 PLP2-TM, SARS-CoV PLpro-TM, MERS-CoV PLpro-TM and PEDV PLP2-TM were transfected into HEK293T cells. As the positive control for induction of autophagy, HEK293T cells were treated with complete medium supplemented with 400 nmol/L Rapamycin for 6 h. The cells were then lysed for Western blotting analysis using a rabbit anti-LC3 antibody to detect endogenous LC3 expression (top panel in Fig. 3A). The whole cell lysate (WCL) was blotted using anti-V5 antibodies to evaluate expression of PLP2 (PLpro)-TM (middle panel in Fig. 3A), and β-Actin was detected in whole cell lysate (WCL) as a loading control (bottom panel in Fig. 3A). (B) PLP2-TM-V5 and the catalytic mutants (C1678A, H1836A and D1849A) as showed in Fig. 1 A were transfected respectively into HEK2923T, and induction of autophagy was detected as described in Fig. 3A
    Figure Legend Snippet: Induction of autophagy by various coronaviral PLPs that is independent on the protease activity . (A) The plasmids of HCoV-NL63 PLP2-TM, SARS-CoV PLpro-TM, MERS-CoV PLpro-TM and PEDV PLP2-TM were transfected into HEK293T cells. As the positive control for induction of autophagy, HEK293T cells were treated with complete medium supplemented with 400 nmol/L Rapamycin for 6 h. The cells were then lysed for Western blotting analysis using a rabbit anti-LC3 antibody to detect endogenous LC3 expression (top panel in Fig. 3A). The whole cell lysate (WCL) was blotted using anti-V5 antibodies to evaluate expression of PLP2 (PLpro)-TM (middle panel in Fig. 3A), and β-Actin was detected in whole cell lysate (WCL) as a loading control (bottom panel in Fig. 3A). (B) PLP2-TM-V5 and the catalytic mutants (C1678A, H1836A and D1849A) as showed in Fig. 1 A were transfected respectively into HEK2923T, and induction of autophagy was detected as described in Fig. 3A

    Techniques Used: Activity Assay, Transfection, Positive Control, Western Blot, Expressing

    PLP2-TM activates autophagosome formation but prevents its fusion with lysosomes . (A) HEK293T cells were transfected with pcDNA3.1 or PLP2-TM. As the positive control for induction of autophagy, HEK293T cells were treated with complete medium supplemented with 400 nmol/L Rapamycin for 6 h. At 48 h post-transfection, the levels of endogenous autophagic substrate p62, LC3 protein and PLP2-TM were determined using Western blot analysis. Beta-actin expression was examined as a protein loading control. (B) Diagram of mRFP-GFP-LC3 structure and the principle for probing autophagy flux with mRFP-GFP-LC3 construct. The GFP signal was easily quenched in autolysosome as the acidic pH lysosomal background because of lysosomal hydrolysis, while mRFP fluorescence existed in the acidic pH background. The merged yellow signal GFP + mRFP + was visualized using a confocal microscope in the autophagosomes and GFP - mRFP + signal was visualized using a confocal microscope in the autolysosomes as described previously (Tang et al., 2013 ). (C) PLP2-TM activates autophagosome formation but blocks its fusion with lysosomes. HEK293T cells were co-transfected with mRFP-GFP-LC3 and PLP2-TM. As the positive control for induction of autophagy, HEK293T cells were transfected with mRFP-GFP-LC3 and then treated with complete medium supplemented with 400 nmol/L Rapamycin for 6 h. For the inhibition of autolysosome maturation, HEK293T cells were transfected with the plasmids of mRFP-GFP-LC3 and then treated with complete medium supplemented with 50 µmol/L CQ for 6 h. At 48 h post-transfection, the cells were fixed and assessed for GFP and mRFP fluorescence. Based on differential pH sensitivity of mRFP and GFP, the mRFP-GFP-LC3 probe differentiates between early, nonacidified autophagosomes (red + green + , yellow in merged images) from acidified, degradative autolysosomes (red + green − , red in merged images)
    Figure Legend Snippet: PLP2-TM activates autophagosome formation but prevents its fusion with lysosomes . (A) HEK293T cells were transfected with pcDNA3.1 or PLP2-TM. As the positive control for induction of autophagy, HEK293T cells were treated with complete medium supplemented with 400 nmol/L Rapamycin for 6 h. At 48 h post-transfection, the levels of endogenous autophagic substrate p62, LC3 protein and PLP2-TM were determined using Western blot analysis. Beta-actin expression was examined as a protein loading control. (B) Diagram of mRFP-GFP-LC3 structure and the principle for probing autophagy flux with mRFP-GFP-LC3 construct. The GFP signal was easily quenched in autolysosome as the acidic pH lysosomal background because of lysosomal hydrolysis, while mRFP fluorescence existed in the acidic pH background. The merged yellow signal GFP + mRFP + was visualized using a confocal microscope in the autophagosomes and GFP - mRFP + signal was visualized using a confocal microscope in the autolysosomes as described previously (Tang et al., 2013 ). (C) PLP2-TM activates autophagosome formation but blocks its fusion with lysosomes. HEK293T cells were co-transfected with mRFP-GFP-LC3 and PLP2-TM. As the positive control for induction of autophagy, HEK293T cells were transfected with mRFP-GFP-LC3 and then treated with complete medium supplemented with 400 nmol/L Rapamycin for 6 h. For the inhibition of autolysosome maturation, HEK293T cells were transfected with the plasmids of mRFP-GFP-LC3 and then treated with complete medium supplemented with 50 µmol/L CQ for 6 h. At 48 h post-transfection, the cells were fixed and assessed for GFP and mRFP fluorescence. Based on differential pH sensitivity of mRFP and GFP, the mRFP-GFP-LC3 probe differentiates between early, nonacidified autophagosomes (red + green + , yellow in merged images) from acidified, degradative autolysosomes (red + green − , red in merged images)

    Techniques Used: Transfection, Positive Control, Western Blot, Expressing, Construct, Fluorescence, Microscopy, Inhibition

    PLP2-TM is a novel autophagy-inducing protein encoded by coronavirus . (A) Schematic diagram illustrating coronavirus NL63 genome, polyprotein (pp) 1a/b, the predicted processing of replicase polyproteins (pp) to nsp’s. The domains, including the predicted transmembrane (TM), in nsp3, and the membrane anchored-PLP2 construct (PLP2-TM) that were used in this study are indicated. (B) PLP2-TM is a novel autophagy-inducing protein encoded by coronavirus. The plasmid of HCoV-NL63 PLP2-TM-V5 was co-transfected with pcDNA3.1-eGFP-LC3B into HEK2923T cells. As the positive control for induction of autophagy, HEK293T cells were transfected with eGFP-LC3B for 48 h and then treated with complete medium supplemented with 400 nmol/L Rapamycin for 6 h. The immunofluoresence of the cells were detected using a confocal microscope after stained with anti-V5-tagged primary antibody, followed by being stained with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody. The localization of eGFP-LC3B positive autophagosome accumulation (green) and the V5-tagged PLPs products (anti-V5, red) was visualized using a confocal microscope. (C) Quantification of cells displaying eGFP-LC3B puncta in PLP2-TM transfected cells. HEK 293T cells were transiently co-transfected with eGFP-LC3B and PLP2-TM-expression constructs. Forty-eight hours later, cells with eGFP-LC3B puncta formation were quantificated under a fluorescence confocal microscope. Three random fields, each containing at least 80 cells, were counted. Results from one representative experiment are shown in Fig. 1B. (D) PLP2-TM induces autophagosome-like structures detected using a transmission electron microscope. HEK293T cells were transfected with pcDNA3.1 or PLP2-TM-V5 for 48 h and then cells were analyzed for autophagosome formation using a transmission electron microscope. The cells treated by 400 nmol/L Rapamycin were analyzed to serve as a positive control for induction of autophagy. Red arrows indicate representative autophagosome-like structures. N indicates the cellular nuclear. Scale bar indicates 500 nm
    Figure Legend Snippet: PLP2-TM is a novel autophagy-inducing protein encoded by coronavirus . (A) Schematic diagram illustrating coronavirus NL63 genome, polyprotein (pp) 1a/b, the predicted processing of replicase polyproteins (pp) to nsp’s. The domains, including the predicted transmembrane (TM), in nsp3, and the membrane anchored-PLP2 construct (PLP2-TM) that were used in this study are indicated. (B) PLP2-TM is a novel autophagy-inducing protein encoded by coronavirus. The plasmid of HCoV-NL63 PLP2-TM-V5 was co-transfected with pcDNA3.1-eGFP-LC3B into HEK2923T cells. As the positive control for induction of autophagy, HEK293T cells were transfected with eGFP-LC3B for 48 h and then treated with complete medium supplemented with 400 nmol/L Rapamycin for 6 h. The immunofluoresence of the cells were detected using a confocal microscope after stained with anti-V5-tagged primary antibody, followed by being stained with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody. The localization of eGFP-LC3B positive autophagosome accumulation (green) and the V5-tagged PLPs products (anti-V5, red) was visualized using a confocal microscope. (C) Quantification of cells displaying eGFP-LC3B puncta in PLP2-TM transfected cells. HEK 293T cells were transiently co-transfected with eGFP-LC3B and PLP2-TM-expression constructs. Forty-eight hours later, cells with eGFP-LC3B puncta formation were quantificated under a fluorescence confocal microscope. Three random fields, each containing at least 80 cells, were counted. Results from one representative experiment are shown in Fig. 1B. (D) PLP2-TM induces autophagosome-like structures detected using a transmission electron microscope. HEK293T cells were transfected with pcDNA3.1 or PLP2-TM-V5 for 48 h and then cells were analyzed for autophagosome formation using a transmission electron microscope. The cells treated by 400 nmol/L Rapamycin were analyzed to serve as a positive control for induction of autophagy. Red arrows indicate representative autophagosome-like structures. N indicates the cellular nuclear. Scale bar indicates 500 nm

    Techniques Used: Construct, Plasmid Preparation, Transfection, Positive Control, Microscopy, Staining, Expressing, Fluorescence, Transmission Assay

    22) Product Images from "Rap1 Promotes Multiple Pancreatic Islet Cell Functions and Signals through Mammalian Target of Rapamycin Complex 1 to Enhance Proliferation *"

    Article Title: Rap1 Promotes Multiple Pancreatic Islet Cell Functions and Signals through Mammalian Target of Rapamycin Complex 1 to Enhance Proliferation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.069112

    Activation of Rap1 promotes GSIS in a rapamycin-insensitive manner. Rat pancreatic islets were isolated and treated with adenoviruses expressing GFP or Rap1A-63E the day before the day of the assay and treated with dimethyl sulfoxide alone or rapamycin.
    Figure Legend Snippet: Activation of Rap1 promotes GSIS in a rapamycin-insensitive manner. Rat pancreatic islets were isolated and treated with adenoviruses expressing GFP or Rap1A-63E the day before the day of the assay and treated with dimethyl sulfoxide alone or rapamycin.

    Techniques Used: Activation Assay, Isolation, Expressing

    23) Product Images from "Regulation of TORC1 by Rag GTPases in nutrient response"

    Article Title: Regulation of TORC1 by Rag GTPases in nutrient response

    Journal: Nature cell biology

    doi: 10.1038/ncb1753

    Relationship between Rag and components of the TOR pathway (a) Rag acts through TORC1 to regulate S6K phosphorylation. transfected with constructs as indicated. Co-expression of 600 ng of mTOR kinase dead (mTOR KD) construct or rapamycin treatment (20 nM, 30 min) abolished the effect of RagA Q66L and RagC S75N on S6K phosphorylation. Protein level of mTOR KD was determined by immunoblotting with anti-mTOR antibody. Molecular weights of markers are indicated on the right. (b) RagA/RagC and TSC1/TSC2 independently regulate S6K phosphorylation. HEK293 cells were transfected with 200 ng of each Rag and/or TSC constructs as indicated. Amino acid starvation for 1 hour (−AA) is indicated. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated. (c) TSC2 and RagA/B independently affect S6K phosphorylation. 20 ng of HA-S6K was transfected into HeLa cells with or without RNAi against human TSC2, RagA and RagB as indicated. (d) RagA T21N and RagC Q120L do not block the Rheb-induced S6K phosphorylation. RagA T21N and RagC Q120L (200 ng each) were transfected into HEK293 cells with or without Rheb construct (20 ng). S6K was included in the co-transfection. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated.
    Figure Legend Snippet: Relationship between Rag and components of the TOR pathway (a) Rag acts through TORC1 to regulate S6K phosphorylation. transfected with constructs as indicated. Co-expression of 600 ng of mTOR kinase dead (mTOR KD) construct or rapamycin treatment (20 nM, 30 min) abolished the effect of RagA Q66L and RagC S75N on S6K phosphorylation. Protein level of mTOR KD was determined by immunoblotting with anti-mTOR antibody. Molecular weights of markers are indicated on the right. (b) RagA/RagC and TSC1/TSC2 independently regulate S6K phosphorylation. HEK293 cells were transfected with 200 ng of each Rag and/or TSC constructs as indicated. Amino acid starvation for 1 hour (−AA) is indicated. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated. (c) TSC2 and RagA/B independently affect S6K phosphorylation. 20 ng of HA-S6K was transfected into HeLa cells with or without RNAi against human TSC2, RagA and RagB as indicated. (d) RagA T21N and RagC Q120L do not block the Rheb-induced S6K phosphorylation. RagA T21N and RagC Q120L (200 ng each) were transfected into HEK293 cells with or without Rheb construct (20 ng). S6K was included in the co-transfection. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated.

    Techniques Used: Transfection, Construct, Expressing, Blocking Assay, Cotransfection

    24) Product Images from "Ectopic NGAL expression can alter sensitivity of breast cancer cells to EGFR, Bcl-2, CaM-K inhibitors and the plant natural product berberine"

    Article Title: Ectopic NGAL expression can alter sensitivity of breast cancer cells to EGFR, Bcl-2, CaM-K inhibitors and the plant natural product berberine

    Journal: Cell Cycle

    doi: 10.4161/cc.22786

    Figure 3. Sensitivity of NGAL- and pLXSN-infected cells and doxorubicin-resistant MCF-7/Dox R cells to the mTORC1 inhibitor rapamycin, doxorubicin and the combination of doxorubicin and a constant dose of 5 nM rapamycin. Cells were collected and
    Figure Legend Snippet: Figure 3. Sensitivity of NGAL- and pLXSN-infected cells and doxorubicin-resistant MCF-7/Dox R cells to the mTORC1 inhibitor rapamycin, doxorubicin and the combination of doxorubicin and a constant dose of 5 nM rapamycin. Cells were collected and

    Techniques Used: Infection

    25) Product Images from "β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells"

    Article Title: β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135614

    HMB increases MEF2 promoter activity and expression of MEF2 transcription factors. (A-C) The expression levels of MEF2 members were analyzed by western-blot using specific antibodies after 24 h incubation with 25 μM HMB (n = 4). (D) Cells were transiently transfected with a 4xMEF luciferase reported plasmid to evaluate MEF2-dependent transcription. Cells were pre-incubated for 30 min with 10 μM PD98059, 20 μM LY294002, 20 nM rapamycin or 10 nM Torin1, then incubated with 25 μM HMB for 24 h. Inhibitors were maintained during the experiment (n = 12). Results represent means ± SEM. * p
    Figure Legend Snippet: HMB increases MEF2 promoter activity and expression of MEF2 transcription factors. (A-C) The expression levels of MEF2 members were analyzed by western-blot using specific antibodies after 24 h incubation with 25 μM HMB (n = 4). (D) Cells were transiently transfected with a 4xMEF luciferase reported plasmid to evaluate MEF2-dependent transcription. Cells were pre-incubated for 30 min with 10 μM PD98059, 20 μM LY294002, 20 nM rapamycin or 10 nM Torin1, then incubated with 25 μM HMB for 24 h. Inhibitors were maintained during the experiment (n = 12). Results represent means ± SEM. * p

    Techniques Used: Activity Assay, Expressing, Western Blot, Incubation, Transfection, Luciferase, Plasmid Preparation

    Rapamycin and Torin1 effects on neurite outgrowth in presence of HMB. Cells were pre-treated with rapamycin 20nM or Torin1 10 nM and then treated with 25 μM HMB for 48 h. The inhibitor was maintained during the experiment. Results represent means ± SEM (n = 8). * p
    Figure Legend Snippet: Rapamycin and Torin1 effects on neurite outgrowth in presence of HMB. Cells were pre-treated with rapamycin 20nM or Torin1 10 nM and then treated with 25 μM HMB for 48 h. The inhibitor was maintained during the experiment. Results represent means ± SEM (n = 8). * p

    Techniques Used:

    HMB activates mTOR and promotes protein synthesis in Neuro2a cells. (A) Neuro2a cells were pre-treated with rapamycin 20nM or Torin1 10 nM and then were treated with 25 μM HMB for 30 min. Western blot analysis was performed using specific antibodies against phospho- and total-antibodies mTOR. (B) Protein synthesis was measured in Neuro2a cells incubated with 25 μM HMB for 2 hours in the absence (n = 10) or presence of inhibitors of PI3K/Akt (LY294002 20μM), ERK1/2 (PD98059 10μM) or mTOR (rapamycin 20nM). Pretreatment of inhibitors occurred as in Fig 2 . Results represent means ± SEM (n = 4). * p
    Figure Legend Snippet: HMB activates mTOR and promotes protein synthesis in Neuro2a cells. (A) Neuro2a cells were pre-treated with rapamycin 20nM or Torin1 10 nM and then were treated with 25 μM HMB for 30 min. Western blot analysis was performed using specific antibodies against phospho- and total-antibodies mTOR. (B) Protein synthesis was measured in Neuro2a cells incubated with 25 μM HMB for 2 hours in the absence (n = 10) or presence of inhibitors of PI3K/Akt (LY294002 20μM), ERK1/2 (PD98059 10μM) or mTOR (rapamycin 20nM). Pretreatment of inhibitors occurred as in Fig 2 . Results represent means ± SEM (n = 4). * p

    Techniques Used: Western Blot, Incubation

    26) Product Images from "TSC1 and TSC2 regulate cilia length and canonical Hedgehog signaling via different mechanisms"

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

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-018-2761-8

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

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

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

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

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

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

    27) Product Images from "Translational control of SCL-isoform expression in hematopoietic lineage choice"

    Article Title: Translational control of SCL-isoform expression in hematopoietic lineage choice

    Journal: Genes & Development

    doi: 10.1101/gad.251903

    Translational controlled differential expression of SCL protein isoforms during erythroid differentiation. ( A ) Erythroid differentiation in HD3 erythroblasts was induced by temperature shift (37°C–42°C for 12 h) to inactivate ts-v-erbB . ( Left ) Rapamycin was added 6 h after temperature shift where indicated. ( Right ) Expression of individual SCL isoforms from sequential translation initiation codons revealed by 5′ deletion of chicken SCL cDNA (termed A–D) in COS-1 cells. SCL-isoform expression was quantified by densitometry of X-ray films and normalized values are depicted below. For immunodetection, specific antiserum against C terminus of chicken SCL was used. ( B ) Human TF-1 or HEL cells were treated for 4–48 h with Epo or DMSO, respectively, to induce erythroid differentiation. 2-Amino purine (2AP) was added where indicated. Immunodetection of SCL in total cell extracts of TF-1 (data shown for 0 and 4 h postinduction) or HEL cells using a human SCL C terminus-specific antiserum is shown. In HEL cells, the SCL-D isoform was obscured by a nonspecific band and was therefore omitted. ( C ) Transiently transfected COS-1 cells with HA-tagged human SCL cDNA ( left panel), and N-terminal-SCL deletion constructs (A–D, right panel). Cells were treated with rapamycin (Rap), or mock (− −) treated for 24 h where indicated. For immunodetection of SCL, an HA-immunotag-specific antiserum was used. SCL protein bands (▸), an alternative CUG (▹), and nonspecific/unidentified bands (*) are indicated. ( D ) Representation of the conserved vertebrate scl structure and translation initiation sites. Numbering of exons follows the nomenclature for the human scl ).
    Figure Legend Snippet: Translational controlled differential expression of SCL protein isoforms during erythroid differentiation. ( A ) Erythroid differentiation in HD3 erythroblasts was induced by temperature shift (37°C–42°C for 12 h) to inactivate ts-v-erbB . ( Left ) Rapamycin was added 6 h after temperature shift where indicated. ( Right ) Expression of individual SCL isoforms from sequential translation initiation codons revealed by 5′ deletion of chicken SCL cDNA (termed A–D) in COS-1 cells. SCL-isoform expression was quantified by densitometry of X-ray films and normalized values are depicted below. For immunodetection, specific antiserum against C terminus of chicken SCL was used. ( B ) Human TF-1 or HEL cells were treated for 4–48 h with Epo or DMSO, respectively, to induce erythroid differentiation. 2-Amino purine (2AP) was added where indicated. Immunodetection of SCL in total cell extracts of TF-1 (data shown for 0 and 4 h postinduction) or HEL cells using a human SCL C terminus-specific antiserum is shown. In HEL cells, the SCL-D isoform was obscured by a nonspecific band and was therefore omitted. ( C ) Transiently transfected COS-1 cells with HA-tagged human SCL cDNA ( left panel), and N-terminal-SCL deletion constructs (A–D, right panel). Cells were treated with rapamycin (Rap), or mock (− −) treated for 24 h where indicated. For immunodetection of SCL, an HA-immunotag-specific antiserum was used. SCL protein bands (▸), an alternative CUG (▹), and nonspecific/unidentified bands (*) are indicated. ( D ) Representation of the conserved vertebrate scl structure and translation initiation sites. Numbering of exons follows the nomenclature for the human scl ).

    Techniques Used: Expressing, Immunodetection, Transfection, Construct

    28) Product Images from "Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 ▿Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 ▿ †"

    Article Title: Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 ▿Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01740-08

    Epistatic analysis of ptc1 ). (B) Rapamycin and caffeine sensitivity of diverse mutants in genes relevant in the TOR pathway in the presence (+) or the absence of PTC1 (−). Cultures were spotted on YPD plates containing the indicated concentrations of the drugs, and growth was monitored after 2 days. WT, wild type.
    Figure Legend Snippet: Epistatic analysis of ptc1 ). (B) Rapamycin and caffeine sensitivity of diverse mutants in genes relevant in the TOR pathway in the presence (+) or the absence of PTC1 (−). Cultures were spotted on YPD plates containing the indicated concentrations of the drugs, and growth was monitored after 2 days. WT, wild type.

    Techniques Used:

    Lack of Ptc1 affects Npr1 levels and phosphorylation state. Wild-type BY4741 (WT) and its ptc1 (MAR143) or sit4 derivative were transformed with plasmid pEJ23, which carries an N-terminally HA-tagged version of Npr1. Cells were incubated for 30 min with 100 ng/ml rapamycin or vehicle, and extracts were prepared. Thirty micrograms of protein [90 μg in the case of ptc1 cells, denoted ptc1 (3x)] was electrophoresed, transferred, and incubated with anti-HA monoclonal antibodies. Faster-migrating bands correspond to the dephosphorylated forms of Npr1.
    Figure Legend Snippet: Lack of Ptc1 affects Npr1 levels and phosphorylation state. Wild-type BY4741 (WT) and its ptc1 (MAR143) or sit4 derivative were transformed with plasmid pEJ23, which carries an N-terminally HA-tagged version of Npr1. Cells were incubated for 30 min with 100 ng/ml rapamycin or vehicle, and extracts were prepared. Thirty micrograms of protein [90 μg in the case of ptc1 cells, denoted ptc1 (3x)] was electrophoresed, transferred, and incubated with anti-HA monoclonal antibodies. Faster-migrating bands correspond to the dephosphorylated forms of Npr1.

    Techniques Used: Transformation Assay, Plasmid Preparation, Incubation

    Global transcriptional response analysis to rapamycin in wild-type (WT) and ptc1 cells. (A) Venn diagram showing the number of genes whose expression was considered to be induced (top) or repressed (bottom) by rapamycin in wild-type ptc1 cells for a set of 4,677 genes, with valid data for both strains. (B) Plots of the log 2 values for the changes in the level of expression induced by rapamycin in both wild-type (open circles) and ptc1 strains for the 150 most upregulated (top) and 150 most downregulated (bottom) genes in the wild-type strain. The expression values for the ptc1 ), ribosomal proteins (open squares), and others (closed triangles).
    Figure Legend Snippet: Global transcriptional response analysis to rapamycin in wild-type (WT) and ptc1 cells. (A) Venn diagram showing the number of genes whose expression was considered to be induced (top) or repressed (bottom) by rapamycin in wild-type ptc1 cells for a set of 4,677 genes, with valid data for both strains. (B) Plots of the log 2 values for the changes in the level of expression induced by rapamycin in both wild-type (open circles) and ptc1 strains for the 150 most upregulated (top) and 150 most downregulated (bottom) genes in the wild-type strain. The expression values for the ptc1 ), ribosomal proteins (open squares), and others (closed triangles).

    Techniques Used: Expressing

    Phosphorylation pattern of Tip41 in Ptc1-deficient cells. The upper panel shows wild-type BY4741 (WT) and ptc1 cells transformed with plasmid pEJ120, which were incubated for 30 min with 100 ng/ml rapamycin (Rap) or vehicle. Extracts were prepared for isoelectrofocusing as described in Materials and Methods. The first dimension was run using Immobiline DryStrip (pH 4 to 7) strips, and the second dimension was performed in SDS-10% polyacrylamide gels. Gels were transferred to membranes, and Tip41 was detected using anti-HA antibodies. Only the relevant region of the immunoblot is shown, and the different Tip41 forms are labeled (A to I). pHs are indicated on the top, and the molecular mass standard is on the right. The lower panel shows the vol% parameter (i.e., relative volume of a spot) for each spot, calculated using Melanie 7.0 software. The means ± standard errors from the means from three independent experiments are represented. The intensity of spots G, F, and E in the ptc1 mutant was too low to be integrated.
    Figure Legend Snippet: Phosphorylation pattern of Tip41 in Ptc1-deficient cells. The upper panel shows wild-type BY4741 (WT) and ptc1 cells transformed with plasmid pEJ120, which were incubated for 30 min with 100 ng/ml rapamycin (Rap) or vehicle. Extracts were prepared for isoelectrofocusing as described in Materials and Methods. The first dimension was run using Immobiline DryStrip (pH 4 to 7) strips, and the second dimension was performed in SDS-10% polyacrylamide gels. Gels were transferred to membranes, and Tip41 was detected using anti-HA antibodies. Only the relevant region of the immunoblot is shown, and the different Tip41 forms are labeled (A to I). pHs are indicated on the top, and the molecular mass standard is on the right. The lower panel shows the vol% parameter (i.e., relative volume of a spot) for each spot, calculated using Melanie 7.0 software. The means ± standard errors from the means from three independent experiments are represented. The intensity of spots G, F, and E in the ptc1 mutant was too low to be integrated.

    Techniques Used: Transformation Assay, Plasmid Preparation, Incubation, Labeling, Software, Mutagenesis

    Sensitivity to rapamycin and caffeine of the diverse yeast type 2C protein phosphatase mutants. (A) Wild-type strain BY4741 and the different ptc mutants were spotted onto YPD plates containing the indicated concentrations of rapamycin or caffeine. Growth was monitored after 60 h of incubation at 28°C. (B) The BY4741 strain and its ptc1 derivative were transformed with an empty centromeric YCplac33 vector (YCp-Ø) or with the wild type or a catalytically inactive form of Ptc1 ( PTC1 [D58N] ) cloned in either centromeric (YCp) or high-copy-number (YEp) vectors. Cells were spotted as described above and incubated for 48 h.
    Figure Legend Snippet: Sensitivity to rapamycin and caffeine of the diverse yeast type 2C protein phosphatase mutants. (A) Wild-type strain BY4741 and the different ptc mutants were spotted onto YPD plates containing the indicated concentrations of rapamycin or caffeine. Growth was monitored after 60 h of incubation at 28°C. (B) The BY4741 strain and its ptc1 derivative were transformed with an empty centromeric YCplac33 vector (YCp-Ø) or with the wild type or a catalytically inactive form of Ptc1 ( PTC1 [D58N] ) cloned in either centromeric (YCp) or high-copy-number (YEp) vectors. Cells were spotted as described above and incubated for 48 h.

    Techniques Used: Incubation, Transformation Assay, Plasmid Preparation, Clone Assay

    Decreased response of diverse NCR-sensitive genes to rapamycin treatment. (A) The indicated constructs were introduced into wild-type BY4741 (WT) and its ptc1 derivative, and cells were treated with 200 ng/ml rapamycin (Rap) (open bars) for 60 (for GAP1 , GLN1 , and GDH1 promoters) or 90 min (for MEP1 ). Control cells (closed bars) received only the solvent. β-Galactosidase activity was measured as indicated in the text. Data are means ± standard errors of the means from six independent clones. (B) RT-PCR experiments were performed using oligonucleotides specific for the indicated genes (see Materials and Methods). Amplification fragments were run on 2% agarose gels. ACT1 is included for comparison.
    Figure Legend Snippet: Decreased response of diverse NCR-sensitive genes to rapamycin treatment. (A) The indicated constructs were introduced into wild-type BY4741 (WT) and its ptc1 derivative, and cells were treated with 200 ng/ml rapamycin (Rap) (open bars) for 60 (for GAP1 , GLN1 , and GDH1 promoters) or 90 min (for MEP1 ). Control cells (closed bars) received only the solvent. β-Galactosidase activity was measured as indicated in the text. Data are means ± standard errors of the means from six independent clones. (B) RT-PCR experiments were performed using oligonucleotides specific for the indicated genes (see Materials and Methods). Amplification fragments were run on 2% agarose gels. ACT1 is included for comparison.

    Techniques Used: Construct, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

    ptc1 mutation impairs rapamycin-induced Gln3 and Msn2 entry into the nucleus. (A) TB123 ( PTC1 GLN3-myc 13 - kanMX ) and AGS39 ( ptc1 GLN3-myc 13 - kanMX ) cells were exposed to 200 ng/ml rapamycin (Rap) or to solvent for 10 min and processed for indirect immunofluorescence using anti-Myc antibodies. Samples also were stained with DAPI to reveal the position of the nuclei (magnification, ×1,000). (B) The upper panel shows wild-type (WT) strain AGS66 and its ptc1 isogenic derivative AGS67, which contain integrated STRE- LacZ reporters. They were treated for 1 h with 200 ng/ml rapamycin (open bars) or solvent (closed bars), and β-galactosidase activity was measured. Data are means ± standard errors of the means from six independent clones. The lower panel shows strains BY4741 (WT) and MAR143 ( ptc1 ) and incubated with 200 ng/ml rapamycin for 15 min. The localization of Msn2 was followed by fluorescence microscopy (magnification, ×1,000).
    Figure Legend Snippet: ptc1 mutation impairs rapamycin-induced Gln3 and Msn2 entry into the nucleus. (A) TB123 ( PTC1 GLN3-myc 13 - kanMX ) and AGS39 ( ptc1 GLN3-myc 13 - kanMX ) cells were exposed to 200 ng/ml rapamycin (Rap) or to solvent for 10 min and processed for indirect immunofluorescence using anti-Myc antibodies. Samples also were stained with DAPI to reveal the position of the nuclei (magnification, ×1,000). (B) The upper panel shows wild-type (WT) strain AGS66 and its ptc1 isogenic derivative AGS67, which contain integrated STRE- LacZ reporters. They were treated for 1 h with 200 ng/ml rapamycin (open bars) or solvent (closed bars), and β-galactosidase activity was measured. Data are means ± standard errors of the means from six independent clones. The lower panel shows strains BY4741 (WT) and MAR143 ( ptc1 ) and incubated with 200 ng/ml rapamycin for 15 min. The localization of Msn2 was followed by fluorescence microscopy (magnification, ×1,000).

    Techniques Used: Mutagenesis, Immunofluorescence, Staining, Activity Assay, Clone Assay, Incubation, Fluorescence, Microscopy

    29) Product Images from "Regnase-1 controls colon epithelial regeneration via regulation of mTOR and purine metabolism"

    Article Title: Regnase-1 controls colon epithelial regeneration via regulation of mTOR and purine metabolism

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1809575115

    Regnase-1 targets to mTOR-signaling pathway. ( A ) Protocol for AOM/DSS-treated colorectal cancer. ( B ) Representative colon tumor and colitis sections from Regnase-1 fl/fl and Regnase-1 ∆IEC mice stained with H E. (Scale bars: 50 μm.) ( C ) Histological scoring for colitis and tumors. Tumor nodules of Regnase-1 fl/fl and Regnase-1 ∆IEC mice were compared. ( D ) Expression levels of Regnase-1 in acute and chronic phase of DSS-induced colitis. ( E ) Percentage change in 2% DSS-treated body weight compared with starting weight in Regnase-1 fl/fl and Regnase-1 ∆IEC mice and rapamycin (10 mg/kg body weight) plus DSS-treated mice. ( F ) Western blot analysis of mTOR-signaling-pathway proteins from Regnase-1 fl/fl and Regnase-1 ∆IEC mice with or without DSS treatment. ( G ) Relative amounts of indicated protein are shown in heat map. Protein amount of Regnase-1 fl/fl without DSS treatment was set as onefold. ( H ) Luciferase assays for 3′-UTRs of mTOR-signaling-pathway proteins. The length of each 3′-UTR is indicated in parentheses. Representative data from two independent animal experiments (three to six mice per group) ( A – G ) and three independent experiments ( H ) are shown. * P
    Figure Legend Snippet: Regnase-1 targets to mTOR-signaling pathway. ( A ) Protocol for AOM/DSS-treated colorectal cancer. ( B ) Representative colon tumor and colitis sections from Regnase-1 fl/fl and Regnase-1 ∆IEC mice stained with H E. (Scale bars: 50 μm.) ( C ) Histological scoring for colitis and tumors. Tumor nodules of Regnase-1 fl/fl and Regnase-1 ∆IEC mice were compared. ( D ) Expression levels of Regnase-1 in acute and chronic phase of DSS-induced colitis. ( E ) Percentage change in 2% DSS-treated body weight compared with starting weight in Regnase-1 fl/fl and Regnase-1 ∆IEC mice and rapamycin (10 mg/kg body weight) plus DSS-treated mice. ( F ) Western blot analysis of mTOR-signaling-pathway proteins from Regnase-1 fl/fl and Regnase-1 ∆IEC mice with or without DSS treatment. ( G ) Relative amounts of indicated protein are shown in heat map. Protein amount of Regnase-1 fl/fl without DSS treatment was set as onefold. ( H ) Luciferase assays for 3′-UTRs of mTOR-signaling-pathway proteins. The length of each 3′-UTR is indicated in parentheses. Representative data from two independent animal experiments (three to six mice per group) ( A – G ) and three independent experiments ( H ) are shown. * P

    Techniques Used: Mouse Assay, Staining, Expressing, Western Blot, Luciferase

    30) Product Images from "Novel Fusion Protein Approach for Efficient High-Throughput Screening of Small Molecule–Mediating Protein-Protein Interactions in Cells and Living Animals"

    Article Title: Novel Fusion Protein Approach for Efficient High-Throughput Screening of Small Molecule–Mediating Protein-Protein Interactions in Cells and Living Animals

    Journal:

    doi: 10.1158/0008-5472.CAN-05-0588

    The fluorescent microscope photograph of 293T cells taken 72 hours post-transfection with constructs using the heterodimerizing proteins FRB and FKBP12 incubated in the presence or absence of rapamycin. The cells were either transfected with one of the
    Figure Legend Snippet: The fluorescent microscope photograph of 293T cells taken 72 hours post-transfection with constructs using the heterodimerizing proteins FRB and FKBP12 incubated in the presence or absence of rapamycin. The cells were either transfected with one of the

    Techniques Used: Microscopy, Transfection, Construct, Incubation

    A, luminometer assay results for the lysates of 293T cells exposed to different concentrations of rapamycin that had been transfected with the vectors of split-RLuc reporter protein–based fusion protein approach containing different linkers (E0,
    Figure Legend Snippet: A, luminometer assay results for the lysates of 293T cells exposed to different concentrations of rapamycin that had been transfected with the vectors of split-RLuc reporter protein–based fusion protein approach containing different linkers (E0,

    Techniques Used: Transfection

    Luminometer assay results for lysates of 293T cells at different time points following exposure to rapamycin. The cells had either been transfected with one of the split-RLuc-based fusion protein vectors, or cotransfected with the two-vector system. The
    Figure Legend Snippet: Luminometer assay results for lysates of 293T cells at different time points following exposure to rapamycin. The cells had either been transfected with one of the split-RLuc-based fusion protein vectors, or cotransfected with the two-vector system. The

    Techniques Used: Transfection, Plasmid Preparation

    A, schematic diagram of a fusion protein–based approach for studying protein-protein interactions using a rapamycin-mediated complementation strategy with either split-RLuc or split-EGFP protein fragments. In this strategy, the NH 2 -terminal and
    Figure Legend Snippet: A, schematic diagram of a fusion protein–based approach for studying protein-protein interactions using a rapamycin-mediated complementation strategy with either split-RLuc or split-EGFP protein fragments. In this strategy, the NH 2 -terminal and

    Techniques Used:

    31) Product Images from "Isoproterenol induces an increase in muscle fiber size by the proliferation of Pax7‐positive cells and in a mTOR‐independent mechanism"

    Article Title: Isoproterenol induces an increase in muscle fiber size by the proliferation of Pax7‐positive cells and in a mTOR‐independent mechanism

    Journal: Cell Biology International

    doi: 10.1002/cbin.11190

    Rapamycin does not inhibit the effects of isoproterenol . Myogenic cells were grown for 24 h and treated with isoproterenol (ISO) 100 nM, or rapamycin 3 µM (RAPA), or with ISO and RAPA concomitantly for the next 48 h (A–H). Control cells were left untreated (A–B). Seventy‐two‐hour cells were labeled with an anti‐sarcomeric‐alpha‐actinin monoclonal antibody (red; A, C, E and G) and the nuclear dye 4,6‐diamino‐2‐phenylindole dyhydrochloride (DAPI) (blue; B, D, F and H). Note the decrease in the size of myotubes when cells were treated with RAPA (E and F). Scale bar in B represents 100 μm. * P
    Figure Legend Snippet: Rapamycin does not inhibit the effects of isoproterenol . Myogenic cells were grown for 24 h and treated with isoproterenol (ISO) 100 nM, or rapamycin 3 µM (RAPA), or with ISO and RAPA concomitantly for the next 48 h (A–H). Control cells were left untreated (A–B). Seventy‐two‐hour cells were labeled with an anti‐sarcomeric‐alpha‐actinin monoclonal antibody (red; A, C, E and G) and the nuclear dye 4,6‐diamino‐2‐phenylindole dyhydrochloride (DAPI) (blue; B, D, F and H). Note the decrease in the size of myotubes when cells were treated with RAPA (E and F). Scale bar in B represents 100 μm. * P

    Techniques Used: Labeling

    32) Product Images from "Role of mammalian target of rapamycin signaling in autophagy and the neurodegenerative process using a senescence accelerated mouse-prone 8 model"

    Article Title: Role of mammalian target of rapamycin signaling in autophagy and the neurodegenerative process using a senescence accelerated mouse-prone 8 model

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2017.4618

    Rapamycin promoted autophagy by inhibiting mTOR signaling. (A) Rapamycin (0.5 µM) administration was able to significantly increase the protein expression levels of LC3-II and beclin 1 (both P
    Figure Legend Snippet: Rapamycin promoted autophagy by inhibiting mTOR signaling. (A) Rapamycin (0.5 µM) administration was able to significantly increase the protein expression levels of LC3-II and beclin 1 (both P

    Techniques Used: Expressing

    Rapamycin treatment may promote cell morphology and alleviate the Tau phosphorylation of neurons from SAMP8. (A) Cell morphology (magnification, ×200). The neurons derived from SAMR1 appear to be in a n ormal state (a, Neurons-SAMR1). The neurons extracted from SAMP8 were in poor state with most processes broken and exhibited ‘bead-like’ changes (indicated by the white arrows) (b, Neurons-SAMP8). When pretreated with 0.5 µM rapamycin, neurons from SAMP8 were partially improved with some smooth and slender processes (indicated by the white arrows; c, Neurons-SAMP8 + 0.5 µM rapamycin). When pretreated with 1.0 µM rapamycin, the cell state was worse than the untreated group and most neurons lacked projections (d, Neurons-SAMP8 + 1.0 µM rapamycin). (B) Western blot analysis was used to investigate Tau phosphorylation. In the neurons-SAMP8 group, the protein expression levels of Tau (pS199) and Tau (pS396) were significantly increased when compared with the control neurons-SAMR1 group. When pretreated with 0.5 µM rapamycin for three days, Tau (pS199) and Tau (pS396) exhibited significantly decreased levels of protein expression when compared with the neurons-SAMP8 group. *P
    Figure Legend Snippet: Rapamycin treatment may promote cell morphology and alleviate the Tau phosphorylation of neurons from SAMP8. (A) Cell morphology (magnification, ×200). The neurons derived from SAMR1 appear to be in a n ormal state (a, Neurons-SAMR1). The neurons extracted from SAMP8 were in poor state with most processes broken and exhibited ‘bead-like’ changes (indicated by the white arrows) (b, Neurons-SAMP8). When pretreated with 0.5 µM rapamycin, neurons from SAMP8 were partially improved with some smooth and slender processes (indicated by the white arrows; c, Neurons-SAMP8 + 0.5 µM rapamycin). When pretreated with 1.0 µM rapamycin, the cell state was worse than the untreated group and most neurons lacked projections (d, Neurons-SAMP8 + 1.0 µM rapamycin). (B) Western blot analysis was used to investigate Tau phosphorylation. In the neurons-SAMP8 group, the protein expression levels of Tau (pS199) and Tau (pS396) were significantly increased when compared with the control neurons-SAMR1 group. When pretreated with 0.5 µM rapamycin for three days, Tau (pS199) and Tau (pS396) exhibited significantly decreased levels of protein expression when compared with the neurons-SAMP8 group. *P

    Techniques Used: Derivative Assay, Western Blot, Expressing

    Rapamycin reduced the protein expression levels of Bcl-2 in primary neurons. (A) Protein expression levels of Bcl-2 were similar in the cortex and hippocampus of the two mouse strains. (B) When neurons-SAMP8 were administered 0.5 µM rapamycin, the Bcl-2 protein expression levels declined significantly. *P
    Figure Legend Snippet: Rapamycin reduced the protein expression levels of Bcl-2 in primary neurons. (A) Protein expression levels of Bcl-2 were similar in the cortex and hippocampus of the two mouse strains. (B) When neurons-SAMP8 were administered 0.5 µM rapamycin, the Bcl-2 protein expression levels declined significantly. *P

    Techniques Used: Expressing

    33) Product Images from "Novel Functional Role of NK3R Expression in the Potentiating Effects on Somatolactin α Autoregulation in grass carp pituitary cells"

    Article Title: Novel Functional Role of NK3R Expression in the Potentiating Effects on Somatolactin α Autoregulation in grass carp pituitary cells

    Journal: Scientific Reports

    doi: 10.1038/srep36102

    Up-regulation of NK3R gene expression by SLα/β in grass carp pituitary cells. (a) Time course of carp SLα (30 nM) and SLβ (30 nM) treatment on NK3R mRNA expression in carp pituitary cells. (b) 24-hr incubation with increasing levels of SLα or SLβ (0.01–100 nM) treatment on NK3R mRNA expression in carp pituitary cells. (c) Effects of 24-hr co-treatment with the PI 3 K inhibitor Ly294002(10 μM) and wortmannin (1 μM), Akt inhibitor HIMOC (10 μM) and mTOR inhibitor rapamycin (20 nM) on SLα (30 nM)- or SLβ (30 nM)-induced NK3R transcript expression in carp pituitary cells. (d) Effects of 24-hr co-treatment with the JAK 2 inhibitor HEX (50 μM), STAT 5 inhibitor IQDMA (50 μM), MEK 1/2 inhibitor U0126 (10 μM) and p38 MAPK inhibitor PD169816 (10 μM) on SLα (30 nM)- or SLβ (30 nM)-induced NK3R mRNA expression. After drug treatment, total RNA was isolated for real-time PCR of NK3R mRNA. In the data present (Mean ± SEM), the groups denoted by different letters represent a significant difference at p
    Figure Legend Snippet: Up-regulation of NK3R gene expression by SLα/β in grass carp pituitary cells. (a) Time course of carp SLα (30 nM) and SLβ (30 nM) treatment on NK3R mRNA expression in carp pituitary cells. (b) 24-hr incubation with increasing levels of SLα or SLβ (0.01–100 nM) treatment on NK3R mRNA expression in carp pituitary cells. (c) Effects of 24-hr co-treatment with the PI 3 K inhibitor Ly294002(10 μM) and wortmannin (1 μM), Akt inhibitor HIMOC (10 μM) and mTOR inhibitor rapamycin (20 nM) on SLα (30 nM)- or SLβ (30 nM)-induced NK3R transcript expression in carp pituitary cells. (d) Effects of 24-hr co-treatment with the JAK 2 inhibitor HEX (50 μM), STAT 5 inhibitor IQDMA (50 μM), MEK 1/2 inhibitor U0126 (10 μM) and p38 MAPK inhibitor PD169816 (10 μM) on SLα (30 nM)- or SLβ (30 nM)-induced NK3R mRNA expression. After drug treatment, total RNA was isolated for real-time PCR of NK3R mRNA. In the data present (Mean ± SEM), the groups denoted by different letters represent a significant difference at p

    Techniques Used: Expressing, Incubation, Isolation, Real-time Polymerase Chain Reaction

    34) Product Images from "Decreased mTOR signalling reduces mitochondrial ROS in brain via accumulation of the telomerase protein TERT within mitochondria"

    Article Title: Decreased mTOR signalling reduces mitochondrial ROS in brain via accumulation of the telomerase protein TERT within mitochondria

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101089

    Rapamycin causes nuclear exclusion of TERT and reduces cellular ROS levels in a TERT-dependent fashion ( A ) Extranuclear TERT levels (as fraction of total cellular anti-TERT immunofluorescence signal) in MCF7 cells treated with the indicated concentrations of rapamycin. Bosutinib (1μM, red bars) was added to inhibit Src kinase. (*P
    Figure Legend Snippet: Rapamycin causes nuclear exclusion of TERT and reduces cellular ROS levels in a TERT-dependent fashion ( A ) Extranuclear TERT levels (as fraction of total cellular anti-TERT immunofluorescence signal) in MCF7 cells treated with the indicated concentrations of rapamycin. Bosutinib (1μM, red bars) was added to inhibit Src kinase. (*P

    Techniques Used: Immunofluorescence

    TERT protein is required for the reduction of H 2 O 2 release from mouse brain mitochondria by rapamycin ( A ) Mouse TERT protein abundance in brain homogenates and isolated mitochondria after 4 months rapamycin treatment. (n=5 per group, t-test, ***p
    Figure Legend Snippet: TERT protein is required for the reduction of H 2 O 2 release from mouse brain mitochondria by rapamycin ( A ) Mouse TERT protein abundance in brain homogenates and isolated mitochondria after 4 months rapamycin treatment. (n=5 per group, t-test, ***p

    Techniques Used: Isolation

    35) Product Images from "Targeting TORC2 in multiple myeloma with a new mTOR kinase inhibitor"

    Article Title: Targeting TORC2 in multiple myeloma with a new mTOR kinase inhibitor

    Journal: Blood

    doi: 10.1182/blood-2010-05-285726

    pp242 is more effective than rapamycin against MM cell lines . (A) Four cell lines were treated with increasing concentrations of rapamycin (x) or pp242 (o) for 72 hours and number of recovered viable cells was assessed. Data presented as percent of control (no inhibitor treatment), mean of triplicate samples. The standard deviation (SD) in all groups was
    Figure Legend Snippet: pp242 is more effective than rapamycin against MM cell lines . (A) Four cell lines were treated with increasing concentrations of rapamycin (x) or pp242 (o) for 72 hours and number of recovered viable cells was assessed. Data presented as percent of control (no inhibitor treatment), mean of triplicate samples. The standard deviation (SD) in all groups was

    Techniques Used: Standard Deviation

    Inhibition of TORC2 activity by pp242 . (A-C) MM cell lines were pre-treated for 30 minutes with increasing concentrations of rapamycin (Rap) or pp242 (pp) and IGF-1 (250 ng/mL) was then added to all cell lines except OPM-2. After 30 minutes additional incubation, protein lysate was immunoblotted for ex-pression of total p70S6kinase (t-P70), phosphorylated P70 (p-P70(T389)), total AKT (t-akt), phosphorylated AKT [p-akt(S473)], total mTOR (t-mtor), phosphorylated mTOR [p-mTOR(S2481)], total NDRG1, phosphorylated NDRG1 (in C), and total ERK or phosphorylated ERK. Arrows in (C) point to the phosphorylated NDRG1 dimer present in control and rapamycin-treated cells. (D) 8226 cells treated with increasing concentrations of pp242 for 1 hour followed by immunoblot for phosphorylated ERK and total ERK. (E) MM1.S and OPM2 cells treated with 100nM of rapamycin for 0, 1, or 24 hours, followed by immunoblot assay for total AKT and S473 phosphorylated AKT.
    Figure Legend Snippet: Inhibition of TORC2 activity by pp242 . (A-C) MM cell lines were pre-treated for 30 minutes with increasing concentrations of rapamycin (Rap) or pp242 (pp) and IGF-1 (250 ng/mL) was then added to all cell lines except OPM-2. After 30 minutes additional incubation, protein lysate was immunoblotted for ex-pression of total p70S6kinase (t-P70), phosphorylated P70 (p-P70(T389)), total AKT (t-akt), phosphorylated AKT [p-akt(S473)], total mTOR (t-mtor), phosphorylated mTOR [p-mTOR(S2481)], total NDRG1, phosphorylated NDRG1 (in C), and total ERK or phosphorylated ERK. Arrows in (C) point to the phosphorylated NDRG1 dimer present in control and rapamycin-treated cells. (D) 8226 cells treated with increasing concentrations of pp242 for 1 hour followed by immunoblot for phosphorylated ERK and total ERK. (E) MM1.S and OPM2 cells treated with 100nM of rapamycin for 0, 1, or 24 hours, followed by immunoblot assay for total AKT and S473 phosphorylated AKT.

    Techniques Used: Inhibition, Activity Assay, Incubation

    36) Product Images from "Beneficial Effects of Early mTORC1 Inhibition after Traumatic Brain Injury"

    Article Title: Beneficial Effects of Early mTORC1 Inhibition after Traumatic Brain Injury

    Journal: Journal of Neurotrauma

    doi: 10.1089/neu.2015.3899

    Rapamycin treatment 1 h after injury improves cognitive function. Animals underwent moderate controlled cortical impact (CCI) injury (1.15 mm depth, 2.4 m/sec velocity) and were treated with a single intraperitoneal injection of rapamycin (1 mg/kg) 1 h after injury. Three days after injury, cognitive function was assessed using the Morris water maze (MWM), measuring latency to platform over a single block of eight equally spaced trials. ( A ) Representative trajectories of animals swimming to the platform in one of four categories were recorded. ( B ) Average latency times across the first four trial blocks did not differ. ( C ) Average latency times in the final four trials showed a significant effect of both injury and treatment (mixed linear model, p
    Figure Legend Snippet: Rapamycin treatment 1 h after injury improves cognitive function. Animals underwent moderate controlled cortical impact (CCI) injury (1.15 mm depth, 2.4 m/sec velocity) and were treated with a single intraperitoneal injection of rapamycin (1 mg/kg) 1 h after injury. Three days after injury, cognitive function was assessed using the Morris water maze (MWM), measuring latency to platform over a single block of eight equally spaced trials. ( A ) Representative trajectories of animals swimming to the platform in one of four categories were recorded. ( B ) Average latency times across the first four trial blocks did not differ. ( C ) Average latency times in the final four trials showed a significant effect of both injury and treatment (mixed linear model, p

    Techniques Used: Size-exclusion Chromatography, Injection, Blocking Assay

    Rapamycin suppresses astrogliosis after controlled cortical impact (CCI). Mice were treated with vehicle (Veh) or rapamycin (Rapa), and analyzed 24 h after CCI or sham surgery. Sections were stained with glial fibrillary acidic protein (GFAP) antibodies (red) and counterstained with 4',6-diamidino-2-phenylindole (DAPI) or Sytox (blue). ( A ) Western blot analysis of GFAP levels normalized to actin in the contralateral (C-side) or lesion side (L-side) after sham surgery or CCI. GFAP levels are elevated in both sides of the CCI-injured hippocampus. ( B ) Low magnification images of the C-side hippocampus after sham or CCI confirm the increase in the number of GFAP-positive cells after CCI. ( C ) Confocal images of area CA1 in the C-side or L-side after sham surgery. ( D ) Confocal images of area CA1 in the C-side or L-side after CCI and Veh or Rapa treatment. ( E ) Confocal images of area CA3 in the C-side or L-side after sham surgery. ( F ) Confocal images of area CA3 in the C-side or L-side after CCI and Veh or Rapa treatment. ( G ) Quantification of the number of GFAP-positive cells per area analyzed in the C-side of the brain in CA1 and CA3 regions. Rapa treatment significantly reduced astrogliosis in the C-side of both hippocampal areas (* p
    Figure Legend Snippet: Rapamycin suppresses astrogliosis after controlled cortical impact (CCI). Mice were treated with vehicle (Veh) or rapamycin (Rapa), and analyzed 24 h after CCI or sham surgery. Sections were stained with glial fibrillary acidic protein (GFAP) antibodies (red) and counterstained with 4',6-diamidino-2-phenylindole (DAPI) or Sytox (blue). ( A ) Western blot analysis of GFAP levels normalized to actin in the contralateral (C-side) or lesion side (L-side) after sham surgery or CCI. GFAP levels are elevated in both sides of the CCI-injured hippocampus. ( B ) Low magnification images of the C-side hippocampus after sham or CCI confirm the increase in the number of GFAP-positive cells after CCI. ( C ) Confocal images of area CA1 in the C-side or L-side after sham surgery. ( D ) Confocal images of area CA1 in the C-side or L-side after CCI and Veh or Rapa treatment. ( E ) Confocal images of area CA3 in the C-side or L-side after sham surgery. ( F ) Confocal images of area CA3 in the C-side or L-side after CCI and Veh or Rapa treatment. ( G ) Quantification of the number of GFAP-positive cells per area analyzed in the C-side of the brain in CA1 and CA3 regions. Rapa treatment significantly reduced astrogliosis in the C-side of both hippocampal areas (* p

    Techniques Used: Mouse Assay, Staining, Western Blot

    Inhibition of mTORC1 signaling by rapamycin. ( A ) Schematic of the timeline of drug treatments and biological processes analyzed after controlled cortical impact (CCI). ( B ) Western blot analysis of normalized pAktT and pS6 levels in the lesion (L) and contralateral (C) side of the hippocampus in vehicle- (Veh) or rapamycin-treated (Rapa) mice 4 h post-CCI or sham surgical procedure. Quantification of pS6 ( C ) and pAktT ( D ) data. Values indicate the average ratio of phosphoryated/total proteins relative to the contralateral side (C-side) of CCI + Veh samples. CCI induced statistically significant pS6 levels only in Veh-treated, but not in Rapa-treated mice (* p
    Figure Legend Snippet: Inhibition of mTORC1 signaling by rapamycin. ( A ) Schematic of the timeline of drug treatments and biological processes analyzed after controlled cortical impact (CCI). ( B ) Western blot analysis of normalized pAktT and pS6 levels in the lesion (L) and contralateral (C) side of the hippocampus in vehicle- (Veh) or rapamycin-treated (Rapa) mice 4 h post-CCI or sham surgical procedure. Quantification of pS6 ( C ) and pAktT ( D ) data. Values indicate the average ratio of phosphoryated/total proteins relative to the contralateral side (C-side) of CCI + Veh samples. CCI induced statistically significant pS6 levels only in Veh-treated, but not in Rapa-treated mice (* p

    Techniques Used: Inhibition, Western Blot, Mouse Assay

    Rapamycin reduces the extent of neuronal damage after controlled cortical impact (CCI). Mice were treated with vehicle (Veh) or rapamycin (Rapa), and analyzed 24 h after CCI. Sections were stained with Fluoro-Jade B and representative confocal images are shown. ( A ) Low magnification images of the hippocampus in the contralateral (C-side) and lesioned (L-side) hippocampus. ( B ) Higher magnification images of areas CA1, CA3, and DG in the L-side after Veh or Rapa treatment. Quantification of the Fluoro-Jade B signal in the pyramidal layer of area CA3 ( C ) and granule layer of the DG ( D ). Rapa treatment significantly reduced the number of damaged neurons in both areas (* p
    Figure Legend Snippet: Rapamycin reduces the extent of neuronal damage after controlled cortical impact (CCI). Mice were treated with vehicle (Veh) or rapamycin (Rapa), and analyzed 24 h after CCI. Sections were stained with Fluoro-Jade B and representative confocal images are shown. ( A ) Low magnification images of the hippocampus in the contralateral (C-side) and lesioned (L-side) hippocampus. ( B ) Higher magnification images of areas CA1, CA3, and DG in the L-side after Veh or Rapa treatment. Quantification of the Fluoro-Jade B signal in the pyramidal layer of area CA3 ( C ) and granule layer of the DG ( D ). Rapa treatment significantly reduced the number of damaged neurons in both areas (* p

    Techniques Used: Mouse Assay, Staining

    37) Product Images from "Marchantin M: a novel inhibitor of proteasome induces autophagic cell death in prostate cancer cells"

    Article Title: Marchantin M: a novel inhibitor of proteasome induces autophagic cell death in prostate cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.285

    Mar treatment induces autophagy in PC3 cells. ( a ) Analysis of LC3B expression and conversion in PC3 cells treated with Mar by western blotting. OD of LC3BII relative to the control (0 h) was quantified as mean±S.D., n =3. ( b ) Electron micrographs showed autophagy induced by Mar. The thick arrows, autolysosome; the thin arrows, autophagosome. Bar, 1 μ m. ( c ) Analysis of LC3B punctate in PCa cells transfected with GFP-LC3B in the presence or absence of Mar and 3-MA. Rapamycin (0.5 μ M) was used as a positive control. PCa cells were pretreated with 5 mM 3-MA for 2 h prior to Mar treatment for 24 h. ( d ) Percentage of cells with punctuate GFP-LC3B to all GFP-positive cells. The cells with over five GFP-LC3B dots were chosen as positive cells. The values represented are means±S.D. for 200 cells each. ** P
    Figure Legend Snippet: Mar treatment induces autophagy in PC3 cells. ( a ) Analysis of LC3B expression and conversion in PC3 cells treated with Mar by western blotting. OD of LC3BII relative to the control (0 h) was quantified as mean±S.D., n =3. ( b ) Electron micrographs showed autophagy induced by Mar. The thick arrows, autolysosome; the thin arrows, autophagosome. Bar, 1 μ m. ( c ) Analysis of LC3B punctate in PCa cells transfected with GFP-LC3B in the presence or absence of Mar and 3-MA. Rapamycin (0.5 μ M) was used as a positive control. PCa cells were pretreated with 5 mM 3-MA for 2 h prior to Mar treatment for 24 h. ( d ) Percentage of cells with punctuate GFP-LC3B to all GFP-positive cells. The cells with over five GFP-LC3B dots were chosen as positive cells. The values represented are means±S.D. for 200 cells each. ** P

    Techniques Used: Expressing, Western Blot, Transfection, Positive Control

    38) Product Images from "Regulation and Role of GLI1 in Cutaneous Squamous Cell Carcinoma Pathogenesis"

    Article Title: Regulation and Role of GLI1 in Cutaneous Squamous Cell Carcinoma Pathogenesis

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2019.01185

    Effects of EGF and/or PI3K, AKT, mTOR, or MEK1/2 inhibition on GLI1 expression level in human cSCC cell lines. qRT-PCR-based analysis of GLI1 expression levels of SCL-1, MET-1 and MET-4 cells (A) after 24 h treatment with 50 nM everolimus, 100 nM rapamycin, 3 µM PI103, 10 µM GDC-0941, 5 µM MK-2206, 20 µM UO126, or 100 nM SCH772984 and (B) after 24 h treatment with the mentioned drugs and concomitant 3 h incubation with 100 ng/µl EGF. GLI1 expression levels were normalized to 18S rRNA gene expression and values of solvent treated controls were set to 1. Results represent mean values + SEM of three independent experiments measured in triplicates. Statistical significance was tested by a nonparametric Mann-Whitney test. * P
    Figure Legend Snippet: Effects of EGF and/or PI3K, AKT, mTOR, or MEK1/2 inhibition on GLI1 expression level in human cSCC cell lines. qRT-PCR-based analysis of GLI1 expression levels of SCL-1, MET-1 and MET-4 cells (A) after 24 h treatment with 50 nM everolimus, 100 nM rapamycin, 3 µM PI103, 10 µM GDC-0941, 5 µM MK-2206, 20 µM UO126, or 100 nM SCH772984 and (B) after 24 h treatment with the mentioned drugs and concomitant 3 h incubation with 100 ng/µl EGF. GLI1 expression levels were normalized to 18S rRNA gene expression and values of solvent treated controls were set to 1. Results represent mean values + SEM of three independent experiments measured in triplicates. Statistical significance was tested by a nonparametric Mann-Whitney test. * P

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Incubation, MANN-WHITNEY

    39) Product Images from "Fission yeast TORC1 regulates phosphorylation of ribosomal S6 proteins in response to nutrients and its activity is inhibited by rapamycin"

    Article Title: Fission yeast TORC1 regulates phosphorylation of ribosomal S6 proteins in response to nutrients and its activity is inhibited by rapamycin

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.060319

    Rapamycin leads to reduction of cell size in wild-type but not tor2 S1837E cells. Exponentially growing cells of 972 (WT) and AN0083 ( tor2 S1837E ) were diluted to OD 595 =0.06 and then incubated in EMM with either DMSO (control) or 200 nM rapamycin at 30°C.
    Figure Legend Snippet: Rapamycin leads to reduction of cell size in wild-type but not tor2 S1837E cells. Exponentially growing cells of 972 (WT) and AN0083 ( tor2 S1837E ) were diluted to OD 595 =0.06 and then incubated in EMM with either DMSO (control) or 200 nM rapamycin at 30°C.

    Techniques Used: Incubation

    Rapamycin inhibits Rps6 phosphorylation by blocking TORC1. Extracts were subjected to immunoblotting with the indicated antibodies. ( A ) Cells of JUp1204 (0 minutes) were added with either 200 nM rapamycin or DMSO (vehicle) and incubated for the indicated
    Figure Legend Snippet: Rapamycin inhibits Rps6 phosphorylation by blocking TORC1. Extracts were subjected to immunoblotting with the indicated antibodies. ( A ) Cells of JUp1204 (0 minutes) were added with either 200 nM rapamycin or DMSO (vehicle) and incubated for the indicated

    Techniques Used: Blocking Assay, Incubation

    40) Product Images from "Thrombopoietin/MPL participates in initiating and maintaining RUNX1-ETO acute myeloid leukemia via PI3K/AKT signaling"

    Article Title: Thrombopoietin/MPL participates in initiating and maintaining RUNX1-ETO acute myeloid leukemia via PI3K/AKT signaling

    Journal: Blood

    doi: 10.1182/blood-2012-03-414649

    Leukemic cells expressing R1E and Mpl are sensitive to THPO signaling through Jak2/Pi3k/Akt. (A) Western blot analysis of signaling proteins activated by Mpl. MIG-R1E/MID (not expressing Mpl) and MIG-R1E/MID-MPL leukemic cells were stimulated with 0, 1, 2.5, 5, and 10 ng/mL THPO after serum starvation. Expression of phospho-Jak2, Jak2, phospho-Stat5, Stat5, phospho-Akt1, Akt1, phospo-Erk1/2, and Erk1/2 was tested by immunoblot analysis. (B) Apoptosis analysis (annexin V(+), 7-AAD-) of MIG-R1E/MID-MPL and MIG-R1E/MID leukemic cells estimated after 48-hour treatment with PBS (black), Thpo (gray), or Thpo and rapamycin (white); P
    Figure Legend Snippet: Leukemic cells expressing R1E and Mpl are sensitive to THPO signaling through Jak2/Pi3k/Akt. (A) Western blot analysis of signaling proteins activated by Mpl. MIG-R1E/MID (not expressing Mpl) and MIG-R1E/MID-MPL leukemic cells were stimulated with 0, 1, 2.5, 5, and 10 ng/mL THPO after serum starvation. Expression of phospho-Jak2, Jak2, phospho-Stat5, Stat5, phospho-Akt1, Akt1, phospo-Erk1/2, and Erk1/2 was tested by immunoblot analysis. (B) Apoptosis analysis (annexin V(+), 7-AAD-) of MIG-R1E/MID-MPL and MIG-R1E/MID leukemic cells estimated after 48-hour treatment with PBS (black), Thpo (gray), or Thpo and rapamycin (white); P

    Techniques Used: Expressing, Western Blot

    Related Articles

    Western Blot:

    Article Title: Inhibition of autophagy-attenuated calcium oxalate crystal-induced renal tubular epithelial cell injury in vivo and in vitro
    Article Snippet: .. Reagents and antibodies Calcium oxalate (Sigma, 455997), 3-methyladenine (Sigma, M9281), rapamycin (Sigma, R0395), 4′,6-diamidino-2-phenylindole (Sigma, D8417), N-acetyl-L-cysteine (Sigma, A7250), catalase (Millipore, 219261-100KU), 2′,7′-dichlorofluorescin diacetate (Sigma, D6883), Lipofectamine 3000 (Invitrogen, L3000008), and rabbit anti-LC3B (Sigma, L7543) for western blot (WB) (1:2000), mouse anti-BECN1 (Cell Signaling Technology, 3495) for WB (1:1000). .. Rabbit anti-LC3B (Abcam, ab51520) (1:1000) and mouse anti-BECN1 (Abcam, ab114071) were used for immunohistochemistry (IHC) (1:500).

    Incubation:

    Article Title: Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus
    Article Snippet: .. Twenty-four hours postinfection with IBV, cells were either fixed immediately using 4% paraformaldehyde or washed four times and incubated for 2 h in FM, HBSS or FM containing 200 nM rapamycin (Sigma, R0395) to induce autophagy. ..

    other:

    Article Title: Autophagy is a protective response to ethanol neurotoxicity
    Article Snippet: The following materials were used: ethanol (Sigma-Aldrich, E7023), bafilomycin A1 (Sigma-Aldrich, B1793), wortmannin (Sigma-Aldrich, W1628), rapamycin (Sigma-Aldrich, R0395), anti-SQSTM1 antibody (Sigma-Aldrich, P0067), anti-actin antibody (Sigma-Aldrich, A5441), anti-LC3 antibody (Medical and Biological Laboratories, PM036), anti-BECN1 antibody (Abcam, ab55878), anti-RPS6K antibody (Cell Signaling Technology, 9202), anti-phospho-RPS6K antibody (Cell Signaling Technology, 9205), MitoTracker Green FM (Invitrogen, M7514) and LysoTracker Red (Invitrogen, L7528).

    Article Title: Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury
    Article Snippet: Rapamycin (Sigma, R0395) was dissolved in absolute ethanol (Fisher Scientific, AC61508), 3-methyladenine (Sigma, M9281) in DMEM (GIBCO, 11966), and Bafilomycin A1 (LC Laboratories, B-1080) in dimethyl sulfoxide (DMSO; Sigma, 472301).

    Article Title: Autophagy activation prevents sevoflurane-induced neurotoxicity in H4 human neuroglioma cells
    Article Snippet: The following materials were used: bafilomycin A1 (Sigma-Aldrich, B1793, St Louis, MO, USA), rapamycin (Sigma-Aldrich, R0395), 3-MA (Sigma-Aldrich, M9281), 4-phenylbutyrate (4-PBA, Sigma-Aldrich, SML0309), anti-C/EBP homologous protein (CHOP) antibody (Cell Signaling Technology, 2895, Danvers, MA, USA), anti-β-actin antibody (Cell Signaling Technology, 4970), anti-SQSTM1/p62 antibody (Medical and Biological Laboratories, PM045, Tokyo, Japan), anti-LC3 antibody (Novus Biologicals, NB100-2220, Littleton, CO, USA), anti-glucose-related protein 78 (GRP78) antibody (Santa Cruz Biotechnology, sc-1050, Santa Cruz, CA, USA), anti-cleaved caspase 3 antibody (Cell Signaling Technology, 9664), HRP-conjugated goat anti-rabbit secondary antibody (Pierce, 31460), and HRP-conjugated goat anti-mouse secondary antibody (Pierce, 31430).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes
    Article Snippet: .. Huntingtin antibody (1:2000, Cat.MAB2166, Chemicon, Billerica, MA, USA); β-actin antibody (1:5000, Cat.A5441, Sigma, St Louis, MO, USA); LC3 antibody (1:1000, Cat.Ab62721, Abcam, University of Cambridge, UK); p62 antibody (1:2000, Cat.PW9860, Enzo Life Science, Lausen, CH, USA); GFAP (1:2000, Cat.c9205, Sigma, St Louis, MO, USA); DAPI (1:10000, Cat. D9564, Sigma, St Louis, MO, USA); GLT-1 (1:3000, Cat. ab58571, Abcam, University of Cambridge, UK); GLAST (1:5000, Cat.ab416, Abcam, University of Cambridge, UK); Cy3-conjugated anti-mouse IgG and HRP-conjugated anti-mouse IgG (1:5000, Cat 715-165-150 and Cat 715-035-1500, Jackson ImmunoResearch, West Grove, PA, USA); rapamycin (Cat. R0395, Sigma, St Louis, MO, USA); 3-MA (3-methyladenine, Cat. M9281, Sigma, St Louis, MO, USA); DHK (dihydrokainate, Cat.D1064, Sigma, St Louis, MO, USA); and 3 H]glutamate (Cat.NET1082250UC, Perkin Elmer, Waltham, MA, USA) were used in this study. ..

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    Millipore rapamycin induction
    <t>Rapamycin</t> treatment does not interfere with ciliogenesis, cilium maintenance and cilium disassembly (A) GFP-PCM1-FKBP restores ciliogenesis defects of satellite-less IMCD3 PCM1 KO cells. IMCD3 PCM1 KO cells stably expressing GFP-PCM1-FKBP and HA-Kif5b-FRB and IMCD3 PCM1 KO cells were serum-starved for 48 h and percentage of ciliated cells was determined by staining for acetylated-tubulin. (B) Validation of IMCD3 perihperal cells for expression of HA-Kif5b and GFP-PCM1-FKBP. Control and rapamycin- induced IMCD3 peripheral cells were stained for GFP, HA, g-tubulin and DAPI. (C) Rapamycin treatment by itself in control IMCD3 cells does not affect the efficiency of ciliogenesis and cilium maintenance. The experiments were performed in IMCD3 cells following the experimental outline for ciliogenesis and maintenance experiment and the fraction of ciliated cells were quantified after 48 h of serum starvation for cilium assembly experiment and 24 h after rapamycin treatment for maintenance experiments. Results shown are the mean of two independent experiments±SD (=200 cells/experiment, n.s: not significant). (D) Effect of peripheral satellite clustering on cilium maintenance. Cells were serum-starved for 48 h, treated with rapamycin for 1 h and percentage of ciliated cells was determined over 24 h by staining for acetylated- tubulin. Cells that were not treated with rapamycin were used as a control. Data represent mean value from two experiments per condition, ± SD (***p
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    Rapamycin treatment does not interfere with ciliogenesis, cilium maintenance and cilium disassembly (A) GFP-PCM1-FKBP restores ciliogenesis defects of satellite-less IMCD3 PCM1 KO cells. IMCD3 PCM1 KO cells stably expressing GFP-PCM1-FKBP and HA-Kif5b-FRB and IMCD3 PCM1 KO cells were serum-starved for 48 h and percentage of ciliated cells was determined by staining for acetylated-tubulin. (B) Validation of IMCD3 perihperal cells for expression of HA-Kif5b and GFP-PCM1-FKBP. Control and rapamycin- induced IMCD3 peripheral cells were stained for GFP, HA, g-tubulin and DAPI. (C) Rapamycin treatment by itself in control IMCD3 cells does not affect the efficiency of ciliogenesis and cilium maintenance. The experiments were performed in IMCD3 cells following the experimental outline for ciliogenesis and maintenance experiment and the fraction of ciliated cells were quantified after 48 h of serum starvation for cilium assembly experiment and 24 h after rapamycin treatment for maintenance experiments. Results shown are the mean of two independent experiments±SD (=200 cells/experiment, n.s: not significant). (D) Effect of peripheral satellite clustering on cilium maintenance. Cells were serum-starved for 48 h, treated with rapamycin for 1 h and percentage of ciliated cells was determined over 24 h by staining for acetylated- tubulin. Cells that were not treated with rapamycin were used as a control. Data represent mean value from two experiments per condition, ± SD (***p

    Journal: bioRxiv

    Article Title: Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium

    doi: 10.1101/2020.02.10.941658

    Figure Lengend Snippet: Rapamycin treatment does not interfere with ciliogenesis, cilium maintenance and cilium disassembly (A) GFP-PCM1-FKBP restores ciliogenesis defects of satellite-less IMCD3 PCM1 KO cells. IMCD3 PCM1 KO cells stably expressing GFP-PCM1-FKBP and HA-Kif5b-FRB and IMCD3 PCM1 KO cells were serum-starved for 48 h and percentage of ciliated cells was determined by staining for acetylated-tubulin. (B) Validation of IMCD3 perihperal cells for expression of HA-Kif5b and GFP-PCM1-FKBP. Control and rapamycin- induced IMCD3 peripheral cells were stained for GFP, HA, g-tubulin and DAPI. (C) Rapamycin treatment by itself in control IMCD3 cells does not affect the efficiency of ciliogenesis and cilium maintenance. The experiments were performed in IMCD3 cells following the experimental outline for ciliogenesis and maintenance experiment and the fraction of ciliated cells were quantified after 48 h of serum starvation for cilium assembly experiment and 24 h after rapamycin treatment for maintenance experiments. Results shown are the mean of two independent experiments±SD (=200 cells/experiment, n.s: not significant). (D) Effect of peripheral satellite clustering on cilium maintenance. Cells were serum-starved for 48 h, treated with rapamycin for 1 h and percentage of ciliated cells was determined over 24 h by staining for acetylated- tubulin. Cells that were not treated with rapamycin were used as a control. Data represent mean value from two experiments per condition, ± SD (***p

    Article Snippet: For rapamycin induction experiments, cells were treated with cells were treated with 100 nm or 500 nm rapamycin (Milipore.

    Techniques: Stable Transfection, Expressing, Staining

    Proper satellite distribution is required for pericentrosomal abundance of satellite residents at varying levels Effects of satellite mispositioning on pericentrosomal abundance of select satellite residents. (A) Spatial localization of the quantified proteins at the centrosome. (B) Summary of the results for the changes in the pericentrosomal abundance of the indicated proteins and their associated functions and spatial localizations at the centrosome and cilia. “Localization at peripheral clusters” represent the group of proteins that concentrate with PCM1 at the periphery upon rapamycin addition to Kif5b-expressing cells. (C) HeLa cells co-expressing GFP-PCM1-FKBP with HA-Kif5b-FRB or HA-BICD2-FRB were treated with rapamycin for 1 h followed by fixation at 24 h. Cells that were not treated with rapamycin were processed in parallel as controls. Cells were stained with anti-GFP to identify cells with complete redistribution to the cell periphery or center, anti-g-tubulin to mark the centrosome and antibodies against the indicated proteins. Images represent centrosomes in cells from the same coverslip taken with the same camera settings. DNA was stained by DAPI. Fluorescence intensity at the centrosome was quantified and average means of the levels in control cells were normalized to 1. n=25 cells per experiment. Data represent mean value from two experiments per condition, ± SD (***p

    Journal: bioRxiv

    Article Title: Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium

    doi: 10.1101/2020.02.10.941658

    Figure Lengend Snippet: Proper satellite distribution is required for pericentrosomal abundance of satellite residents at varying levels Effects of satellite mispositioning on pericentrosomal abundance of select satellite residents. (A) Spatial localization of the quantified proteins at the centrosome. (B) Summary of the results for the changes in the pericentrosomal abundance of the indicated proteins and their associated functions and spatial localizations at the centrosome and cilia. “Localization at peripheral clusters” represent the group of proteins that concentrate with PCM1 at the periphery upon rapamycin addition to Kif5b-expressing cells. (C) HeLa cells co-expressing GFP-PCM1-FKBP with HA-Kif5b-FRB or HA-BICD2-FRB were treated with rapamycin for 1 h followed by fixation at 24 h. Cells that were not treated with rapamycin were processed in parallel as controls. Cells were stained with anti-GFP to identify cells with complete redistribution to the cell periphery or center, anti-g-tubulin to mark the centrosome and antibodies against the indicated proteins. Images represent centrosomes in cells from the same coverslip taken with the same camera settings. DNA was stained by DAPI. Fluorescence intensity at the centrosome was quantified and average means of the levels in control cells were normalized to 1. n=25 cells per experiment. Data represent mean value from two experiments per condition, ± SD (***p

    Article Snippet: For rapamycin induction experiments, cells were treated with cells were treated with 100 nm or 500 nm rapamycin (Milipore.

    Techniques: Expressing, Staining, Fluorescence

    Development and validation of the inducible trafficking assay (A) Localization of GFP-PCM1 and GFP-PCM1-FKBP in cells. HeLa cells were transfected with GFP-PCM1 or GFP-PCM1-FKBP, fixed after 24 h and stained for GFP, PCM1 and DAPI. (B) Effects of HA-BICD2 or HA-Kif5b expression on satellite distribution. HeLa cells were transfected with HA-BICD2 or HA-Kif5b, fixed after 24 h and stained for HA, PCM1, g-tubulin and DAPI. (C, D) Validation of GFP-PCM1-FKBP and endogenous PCM1 mispositioning upon rapamycin-induced dimerization in cells. Hela cells co-expressing GFP-PCM1-FKBP with (C) HA-Kif5b-FRB or (D) HA-BICD2- FRB were treated with rapamycin for 1 h, fixed 24 h after transfection and stained for GFP, HA, PCM1 and DAPI. Cells that were not treated with rapamycin were processed in parallel as controls. (E) Representation of partial distribution of satellites upon rapamycin induction. Hela cells co-expressing GFP-PCM1-FKBP with HA-Kif5b-FRB or HA-BICD2-FRB were treated with rapamycin for 1 h, fixed 24 h after transfection and stained for GFP, HA, PCM1 and DAPI. Partial distribution was defined by GFP-PCM1- FKBP signal in the pericentrosomal area in Kif5b-expressing cells and in the region excluding therosomal area in BICD2-expressing cells. (F) Expression of GFP-PCM1- FKBP with HA-Kif5b-FRB or HA-BICD2-FRB and their redistribution upon rapamycin induction do not perturb the microtubule network. Cells were stained for GFP, alpha- tubulin and DAPI. (G) Rapamycin treatment did not perturb satellite distribution in wild- type cells or cells expressing only GFP-PCM1-FKBP. Cells were treated with rapamycin for 1 h, fixed after 24 h and stained for GFP or PCM1, g-tubulin and DAPI. F) Co- expression of GFP-PCM1-FKBP with the constitutively active HA-Kif17 (1-181 a.a.)- FRB targets satellites to the cell periphery where satellite clusters are heterogeneously distributed. Transfected HeLa cells were treated with rapamycin for 1 h, fixed after 24 h and stained for GFP, PCM1, g-tubulin and DAPI. Scale bars = 10 μm, all insets show 4X enlarged centrosomes.

    Journal: bioRxiv

    Article Title: Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium

    doi: 10.1101/2020.02.10.941658

    Figure Lengend Snippet: Development and validation of the inducible trafficking assay (A) Localization of GFP-PCM1 and GFP-PCM1-FKBP in cells. HeLa cells were transfected with GFP-PCM1 or GFP-PCM1-FKBP, fixed after 24 h and stained for GFP, PCM1 and DAPI. (B) Effects of HA-BICD2 or HA-Kif5b expression on satellite distribution. HeLa cells were transfected with HA-BICD2 or HA-Kif5b, fixed after 24 h and stained for HA, PCM1, g-tubulin and DAPI. (C, D) Validation of GFP-PCM1-FKBP and endogenous PCM1 mispositioning upon rapamycin-induced dimerization in cells. Hela cells co-expressing GFP-PCM1-FKBP with (C) HA-Kif5b-FRB or (D) HA-BICD2- FRB were treated with rapamycin for 1 h, fixed 24 h after transfection and stained for GFP, HA, PCM1 and DAPI. Cells that were not treated with rapamycin were processed in parallel as controls. (E) Representation of partial distribution of satellites upon rapamycin induction. Hela cells co-expressing GFP-PCM1-FKBP with HA-Kif5b-FRB or HA-BICD2-FRB were treated with rapamycin for 1 h, fixed 24 h after transfection and stained for GFP, HA, PCM1 and DAPI. Partial distribution was defined by GFP-PCM1- FKBP signal in the pericentrosomal area in Kif5b-expressing cells and in the region excluding therosomal area in BICD2-expressing cells. (F) Expression of GFP-PCM1- FKBP with HA-Kif5b-FRB or HA-BICD2-FRB and their redistribution upon rapamycin induction do not perturb the microtubule network. Cells were stained for GFP, alpha- tubulin and DAPI. (G) Rapamycin treatment did not perturb satellite distribution in wild- type cells or cells expressing only GFP-PCM1-FKBP. Cells were treated with rapamycin for 1 h, fixed after 24 h and stained for GFP or PCM1, g-tubulin and DAPI. F) Co- expression of GFP-PCM1-FKBP with the constitutively active HA-Kif17 (1-181 a.a.)- FRB targets satellites to the cell periphery where satellite clusters are heterogeneously distributed. Transfected HeLa cells were treated with rapamycin for 1 h, fixed after 24 h and stained for GFP, PCM1, g-tubulin and DAPI. Scale bars = 10 μm, all insets show 4X enlarged centrosomes.

    Article Snippet: For rapamycin induction experiments, cells were treated with cells were treated with 100 nm or 500 nm rapamycin (Milipore.

    Techniques: Transfection, Staining, Expressing

    The proximity interactome of PCM1 at the peripheral clusters is enriched for proteins implicated in ciliogenesis and mitosis The proximity PCM1 interactome of satellites were identified using the BioID approach. (A) HEK293T cells were transfected with Myc-BirA*-PCM1-FKBP and induced for peripheral targeting of satellites with rapamycin treatment for 1 h. Cells that were not treated with rapamycin were used as a control. After 18 h biotin incubation, cells were fixed and stained for Myc-BirA*-PCM1-FKBP expression with anti-myc, biotinylated proteins with streptavidin and centrosomes with g-tubulin. DNA was stained with DAPI. Scale bar = 10 μm, cell edges are outlined. (B) Biotinylated proteins from cell lysates from cells expressing Myc-BirA*-PCM1-FKBP (-rapamycin or +rapamycin) were pulled down with streptavidin chromatography and samples were analyzed by SDS-PAGE and western blotting with streptavidin to detect biotinylated proteins and with anti-Cep131 (positive control). IS: initial sample used for streptavidin pulldowns, Beads: Eluted proteins. (C, D) GO-enrichment analysis of the proximity interactors of PCM1 after rapamycin treatment based on their (C) biological processes and (D) cellular compartment. The X-axis represents the log transformed p-value (Fishers exact test) of GO terms. (E) The cilium-associated proteins in the interactome of peripheral satellites were determined based on GO categories and previous work and different ciliogenesis functional modules plotted in the “peripheral PCM1 interaction network using Cytoscape.

    Journal: bioRxiv

    Article Title: Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium

    doi: 10.1101/2020.02.10.941658

    Figure Lengend Snippet: The proximity interactome of PCM1 at the peripheral clusters is enriched for proteins implicated in ciliogenesis and mitosis The proximity PCM1 interactome of satellites were identified using the BioID approach. (A) HEK293T cells were transfected with Myc-BirA*-PCM1-FKBP and induced for peripheral targeting of satellites with rapamycin treatment for 1 h. Cells that were not treated with rapamycin were used as a control. After 18 h biotin incubation, cells were fixed and stained for Myc-BirA*-PCM1-FKBP expression with anti-myc, biotinylated proteins with streptavidin and centrosomes with g-tubulin. DNA was stained with DAPI. Scale bar = 10 μm, cell edges are outlined. (B) Biotinylated proteins from cell lysates from cells expressing Myc-BirA*-PCM1-FKBP (-rapamycin or +rapamycin) were pulled down with streptavidin chromatography and samples were analyzed by SDS-PAGE and western blotting with streptavidin to detect biotinylated proteins and with anti-Cep131 (positive control). IS: initial sample used for streptavidin pulldowns, Beads: Eluted proteins. (C, D) GO-enrichment analysis of the proximity interactors of PCM1 after rapamycin treatment based on their (C) biological processes and (D) cellular compartment. The X-axis represents the log transformed p-value (Fishers exact test) of GO terms. (E) The cilium-associated proteins in the interactome of peripheral satellites were determined based on GO categories and previous work and different ciliogenesis functional modules plotted in the “peripheral PCM1 interaction network using Cytoscape.

    Article Snippet: For rapamycin induction experiments, cells were treated with cells were treated with 100 nm or 500 nm rapamycin (Milipore.

    Techniques: Transfection, Incubation, Staining, Expressing, Chromatography, SDS Page, Western Blot, Positive Control, Transformation Assay, Functional Assay

    Pericentrosomal satellite clustering is required for efficient cilium assembly, maintenance and disassembly (A, B) Effect of peripheral satellite clustering on cilium assembly. Control and rapamycin-treated IMCD3 PCM1 KO peripheral cells stably expressing GFP-PCM1-FKBP and HA-Kif5b-FRB were serum-starved for 48 h and percentage of ciliated cells was determined by staining for acetylated-tubulin in cells with complete satellite redistribution to the periphery as assessed by GFP staining. Representative images of ciliated and unciliated cells with peripheral satellite clustering relative to ciliated control cells. DNA was stained with DAPI. Scale bar, 10 μm (C, D) Quantification of ciliogenesis experiments in (C) IMCD3 PCM1 KO peripheral and (D) IMCD3 peripheral cells. Results shown are the mean of two independent experiments±SD (=50 cells/experiment, **p

    Journal: bioRxiv

    Article Title: Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium

    doi: 10.1101/2020.02.10.941658

    Figure Lengend Snippet: Pericentrosomal satellite clustering is required for efficient cilium assembly, maintenance and disassembly (A, B) Effect of peripheral satellite clustering on cilium assembly. Control and rapamycin-treated IMCD3 PCM1 KO peripheral cells stably expressing GFP-PCM1-FKBP and HA-Kif5b-FRB were serum-starved for 48 h and percentage of ciliated cells was determined by staining for acetylated-tubulin in cells with complete satellite redistribution to the periphery as assessed by GFP staining. Representative images of ciliated and unciliated cells with peripheral satellite clustering relative to ciliated control cells. DNA was stained with DAPI. Scale bar, 10 μm (C, D) Quantification of ciliogenesis experiments in (C) IMCD3 PCM1 KO peripheral and (D) IMCD3 peripheral cells. Results shown are the mean of two independent experiments±SD (=50 cells/experiment, **p

    Article Snippet: For rapamycin induction experiments, cells were treated with cells were treated with 100 nm or 500 nm rapamycin (Milipore.

    Techniques: Stable Transfection, Expressing, Staining

    Expression of LC3 and insulin in the presence or absence of rapamycin, in rat INS-1E insulinoma cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with or without rapamycin (20–80 nM) for 24 h at 37 °C with 5% CO 2 . LC3 and insulin were measured by Western blot (A). The relative amounts of LC3 (B) and insulin (C) were quantified as described in the Methods section. Data represent mean±SD of three experiments. * p

    Journal: Data in Brief

    Article Title: Data of intracellular insulin protein reduced by autophagy in INS-1E cells

    doi: 10.1016/j.dib.2016.07.008

    Figure Lengend Snippet: Expression of LC3 and insulin in the presence or absence of rapamycin, in rat INS-1E insulinoma cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with or without rapamycin (20–80 nM) for 24 h at 37 °C with 5% CO 2 . LC3 and insulin were measured by Western blot (A). The relative amounts of LC3 (B) and insulin (C) were quantified as described in the Methods section. Data represent mean±SD of three experiments. * p

    Article Snippet: 2.2 Treatment with 2-DG, rapamycin, bafilomycin A1, and 3-methyladenine INS-1E cells were cultured in a 37 °C and 5% CO2 incubator in RPMI 1640 medium plus 2% heat-inactivated FBS with 2-DG (5 mM) (Sigma-Aldrich, St. Louis, MO, USA), or with rapamycin (20 to 80 nM) (Calbiochem, San Diego, MO, USA), for 24 h. INS-1E cells were also cultured in a 37 °C and 5% CO2 incubator in RPMI 1640 medium plus 2% heat-inactivated FBS with or without 2-DG (5 mM) or rapamycin, and with or without bafilomycin A1 (100 nM) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 3-methyladenine (10 mM) (Santa Cruz Biotechnology) for 6 h.

    Techniques: Expressing, Incubation, Western Blot

    Expression of cellular insulin following bafilomycin A1 or 3-methyladenine treatment with rapamycin in rat INS-1E insulinoma cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with or without rapamycin (80 nM) for 24 h, and with or without bafilomycin A1 (100 nM) or 3-methyladenine (10 mM) for 6 h at 37 °C with 5% CO 2 . Insulin was measured by Western blot (A) and the relative amounts of insulin (B) were quantified as described in the Methods section. Data represent mean ± SD of three experiments. * p

    Journal: Data in Brief

    Article Title: Data of intracellular insulin protein reduced by autophagy in INS-1E cells

    doi: 10.1016/j.dib.2016.07.008

    Figure Lengend Snippet: Expression of cellular insulin following bafilomycin A1 or 3-methyladenine treatment with rapamycin in rat INS-1E insulinoma cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with or without rapamycin (80 nM) for 24 h, and with or without bafilomycin A1 (100 nM) or 3-methyladenine (10 mM) for 6 h at 37 °C with 5% CO 2 . Insulin was measured by Western blot (A) and the relative amounts of insulin (B) were quantified as described in the Methods section. Data represent mean ± SD of three experiments. * p

    Article Snippet: 2.2 Treatment with 2-DG, rapamycin, bafilomycin A1, and 3-methyladenine INS-1E cells were cultured in a 37 °C and 5% CO2 incubator in RPMI 1640 medium plus 2% heat-inactivated FBS with 2-DG (5 mM) (Sigma-Aldrich, St. Louis, MO, USA), or with rapamycin (20 to 80 nM) (Calbiochem, San Diego, MO, USA), for 24 h. INS-1E cells were also cultured in a 37 °C and 5% CO2 incubator in RPMI 1640 medium plus 2% heat-inactivated FBS with or without 2-DG (5 mM) or rapamycin, and with or without bafilomycin A1 (100 nM) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 3-methyladenine (10 mM) (Santa Cruz Biotechnology) for 6 h.

    Techniques: Expressing, Incubation, Western Blot

    Effect of combinatorial treatment of bortezomib (Bz) and rapamycin (Rm) on Echinococcus cell viability. (A) Effect of Rm and Bz on E . granulosus protoscolex viability at 10 μM for individual drugs and 5 μM or 10 μM of each drug for a combined protocol over 7 days (the drugs were replenished after 3 days). Protoscoleces incubated in culture medium containing 1:1.000 DMSO were used as controls. Each point represents the mean percentage of vital protoscoleces from three different experiments. Asterisks indicate a statistically significant difference (P

    Journal: PLoS ONE

    Article Title: Bortezomib initiates endoplasmic reticulum stress, elicits autophagy and death in Echinococcus granulosus larval stage

    doi: 10.1371/journal.pone.0181528

    Figure Lengend Snippet: Effect of combinatorial treatment of bortezomib (Bz) and rapamycin (Rm) on Echinococcus cell viability. (A) Effect of Rm and Bz on E . granulosus protoscolex viability at 10 μM for individual drugs and 5 μM or 10 μM of each drug for a combined protocol over 7 days (the drugs were replenished after 3 days). Protoscoleces incubated in culture medium containing 1:1.000 DMSO were used as controls. Each point represents the mean percentage of vital protoscoleces from three different experiments. Asterisks indicate a statistically significant difference (P

    Article Snippet: In vitro protoscolex and metacestode treatments were assayed with rapamycin (Rm, Calbiochem, USA) and Bortezomib (Bz, Janssen-Cilag, Germany) dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Incubation

    Reversal of carrageenan-induced hyperalgesic priming by local injection of the mTOR inhibitor rapamycin

    Journal: The journal of pain : official journal of the American Pain Society

    Article Title: Peripheral Administration of Translation Inhibitors Reverses Increased Hyperalgesia in a Model of Chronic Pain in the Rat

    doi: 10.1016/j.jpain.2013.01.779

    Figure Lengend Snippet: Reversal of carrageenan-induced hyperalgesic priming by local injection of the mTOR inhibitor rapamycin

    Article Snippet: Cordycepin 5′-triphosphate sodium salt, λ-carrageenan and prostaglandin E2 (PGE2 ) were purchased from Sigma-Aldrich (St. Louis, MO), and rapamycin from EMD Chemicals (Gibbstown, NJ).

    Techniques: Injection

    Prevention of carrageenan-induced hyperalgesic priming by local injection of the mTOR inhibitor rapamycin

    Journal: The journal of pain : official journal of the American Pain Society

    Article Title: Peripheral Administration of Translation Inhibitors Reverses Increased Hyperalgesia in a Model of Chronic Pain in the Rat

    doi: 10.1016/j.jpain.2013.01.779

    Figure Lengend Snippet: Prevention of carrageenan-induced hyperalgesic priming by local injection of the mTOR inhibitor rapamycin

    Article Snippet: Cordycepin 5′-triphosphate sodium salt, λ-carrageenan and prostaglandin E2 (PGE2 ) were purchased from Sigma-Aldrich (St. Louis, MO), and rapamycin from EMD Chemicals (Gibbstown, NJ).

    Techniques: Injection