Structured Review

Millipore rapamycin
Metabolic profiles of 16 month-old mice on high fat diet 8 months from the beginning of the of treatment with different schedules of <t>rapamycin</t> or metformin. p values: the differences with control group (HFD). Glucose, insulin and triglycerides were determined
Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rapamycin/product/Millipore
Average 99 stars, based on 86 article reviews
Price from $9.99 to $1999.99
rapamycin - by Bioz Stars, 2020-02
99/100 stars

Images

1) 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

2) 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

3) Product Images from "Piecemeal Microautophagy of Nucleus in Saccharomyces cerevisiae"

Article Title: Piecemeal Microautophagy of Nucleus in Saccharomyces cerevisiae

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-08-0483

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

Techniques Used: Labeling

4) Product Images from "Sirolimus and metformin synergistically inhibit hepatocellular carcinoma cell proliferation and improve long-term survival in patients with HCC related to hepatitis B virus induced cirrhosis after liver transplantation"

Article Title: Sirolimus and metformin synergistically inhibit hepatocellular carcinoma cell proliferation and improve long-term survival in patients with HCC related to hepatitis B virus induced cirrhosis after liver transplantation

Journal: Oncotarget

doi: 10.18632/oncotarget.11591

Effects of combined treatment of rapamycin and metformin on cell proliferation Inhibition of cell proliferation was observed in cells treated with either rapamycin or metformin. The most significant inhibition of cell growth was observed in cells treated with both rapamycin and metformin. *P
Figure Legend Snippet: Effects of combined treatment of rapamycin and metformin on cell proliferation Inhibition of cell proliferation was observed in cells treated with either rapamycin or metformin. The most significant inhibition of cell growth was observed in cells treated with both rapamycin and metformin. *P

Techniques Used: Inhibition

Overall survival time curve and disease-free survival time curve in the four groups: Group 1.0: the sirolimus and metformin combination (Sir+Met); Group 2.0: sirolimus monotherapy (Sir); Group 3.0: No Sir with DM; Group 4.0: No Sir without DM
Figure Legend Snippet: Overall survival time curve and disease-free survival time curve in the four groups: Group 1.0: the sirolimus and metformin combination (Sir+Met); Group 2.0: sirolimus monotherapy (Sir); Group 3.0: No Sir with DM; Group 4.0: No Sir without DM

Techniques Used:

Effects of Rapamycin and metformin on the expression of proteins involved in the PI3K/AKT pathway C: control; R: rapamycin; M: metformin; RM: combined rapamycin and metformin. Rapamycin and metformin combination caused a significant decrease in phosphorylated PI3K (P-PI3K), P-AKT, AKT, P-AMPK, AMPK, P-mTOR, mTOR, P-4EBP1, 4EBP1 and S6K levels.
Figure Legend Snippet: Effects of Rapamycin and metformin on the expression of proteins involved in the PI3K/AKT pathway C: control; R: rapamycin; M: metformin; RM: combined rapamycin and metformin. Rapamycin and metformin combination caused a significant decrease in phosphorylated PI3K (P-PI3K), P-AKT, AKT, P-AMPK, AMPK, P-mTOR, mTOR, P-4EBP1, 4EBP1 and S6K levels.

Techniques Used: Expressing

Effects of rapamycin and metformin on tumor growth in vivo rapamycin or metformin alone suppressed tumor growth in vivo . The rapamycin and metformin combination had the most pronounced inhibitory effect compared with monotherapies. *P
Figure Legend Snippet: Effects of rapamycin and metformin on tumor growth in vivo rapamycin or metformin alone suppressed tumor growth in vivo . The rapamycin and metformin combination had the most pronounced inhibitory effect compared with monotherapies. *P

Techniques Used: In Vivo

5) Product Images from "bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway"

Article Title: bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway

Journal: Scientific Reports

doi: 10.1038/srep09287

bFGF activates the Akt/mTOR signaling pathways and inhibits rapamycin-induced protein ubiquitination after 12 h treatment in H9C2 cells. (A) Protein expression and optical density analysis of Ub and SQSTM1/p-62 (Thr269/Ser272) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group in H9C2 cells. (B) Protein expression and the optical density analysis of p-AKT (Ser473) and p-mTOR (Ser2448) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group of H9C2 cells. * P
Figure Legend Snippet: bFGF activates the Akt/mTOR signaling pathways and inhibits rapamycin-induced protein ubiquitination after 12 h treatment in H9C2 cells. (A) Protein expression and optical density analysis of Ub and SQSTM1/p-62 (Thr269/Ser272) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group in H9C2 cells. (B) Protein expression and the optical density analysis of p-AKT (Ser473) and p-mTOR (Ser2448) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group of H9C2 cells. * P

Techniques Used: Expressing

Silencing of p62 partially attenuates the anti-apoptosis effect of bFGF, whereas shRNA against the autophagic machinery Atg7 prolongs the survival of cells co-treated with bFGF and rapamycin. (A, B) Protein expression and optical density analysis of Ub and p62 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si p62: p62 siRNA). (C, D) H9C2 cells (si co: random siRNA, si p62: p62 siRNA) were treated with 100 ng/ml rapamycin, with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells.
Figure Legend Snippet: Silencing of p62 partially attenuates the anti-apoptosis effect of bFGF, whereas shRNA against the autophagic machinery Atg7 prolongs the survival of cells co-treated with bFGF and rapamycin. (A, B) Protein expression and optical density analysis of Ub and p62 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si p62: p62 siRNA). (C, D) H9C2 cells (si co: random siRNA, si p62: p62 siRNA) were treated with 100 ng/ml rapamycin, with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells.

Techniques Used: shRNA, Expressing, Staining, Flow Cytometry, Cytometry

The protective effect of bFGF in I/R is inhibited by rapamycin. (A) The echocardiography analysis of I/R, I/R rats treated with rapamycin, bFGF and bFGF with rapamycin. The left ventricular dimension at end-diastole and end-systole (LVEDd, LVEDs), FS = 100 × (LVEDd – LVEDs)/LVEDd. * P
Figure Legend Snippet: The protective effect of bFGF in I/R is inhibited by rapamycin. (A) The echocardiography analysis of I/R, I/R rats treated with rapamycin, bFGF and bFGF with rapamycin. The left ventricular dimension at end-diastole and end-systole (LVEDd, LVEDs), FS = 100 × (LVEDd – LVEDs)/LVEDd. * P

Techniques Used:

The anti-apoptosis effects of bFGF are related to the inhibition of autophagy, which is induced by rapamycin. (A) H9C2 cells were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells. (B) A bar diagram of the apoptotic cell rate from three separate experiments. ** P
Figure Legend Snippet: The anti-apoptosis effects of bFGF are related to the inhibition of autophagy, which is induced by rapamycin. (A) H9C2 cells were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells. (B) A bar diagram of the apoptotic cell rate from three separate experiments. ** P

Techniques Used: Inhibition, Staining, Flow Cytometry, Cytometry

The cardioprotective effects of bFGF are related to the inhibition of autophagy induced by rapamycin. (A) Immunofluorescence staining results of LC3 (green), in which the nuclei are labeled with Hoechst (blue). (B, C) The protein expression of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. The optical density analysis of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. (D) H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA) were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with TUNEL. (E) Protein expression of LC3II/LC3I ATG-7, Ub, SQSTM1/p-62 (Thr269/Ser272) and cleaved-caspase-3 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA). * P
Figure Legend Snippet: The cardioprotective effects of bFGF are related to the inhibition of autophagy induced by rapamycin. (A) Immunofluorescence staining results of LC3 (green), in which the nuclei are labeled with Hoechst (blue). (B, C) The protein expression of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. The optical density analysis of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. (D) H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA) were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with TUNEL. (E) Protein expression of LC3II/LC3I ATG-7, Ub, SQSTM1/p-62 (Thr269/Ser272) and cleaved-caspase-3 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA). * P

Techniques Used: Inhibition, Immunofluorescence, Staining, Labeling, Expressing, TUNEL Assay

6) Product Images from "Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis"

Article Title: Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-17-0925

Patient-derived SMC lines exhibit hyperactive mTORC1 signaling. (A) Simplified mTORC1 signaling network. (B) Representative western blot for pan-S6K and its T389 phosphorylated form (P-S6K) in SMC lines, and densitometry based quantification expressed as relative to control 120ls-SMCs. Average diameter of cells (C) and change in LC3B-II levels with or without bafilomycin A1 (D) in SMC lines. (E) Western blot for HIF1α (left panel) and densitometry-based quantification (right panel) expressed as relative to 121-SMC control. (F) Densitometry-based quantification of western blots for HIF1α expression in SMC lines in the presence of DMSO (vehicle) or rapamycin, expressed as relative to the DMSO condition for each cell line. Statistics are relative to 120ls-SMC (B, C, D), 121-SMC (E), and DMSO condition (F) (t-test). P values of
Figure Legend Snippet: Patient-derived SMC lines exhibit hyperactive mTORC1 signaling. (A) Simplified mTORC1 signaling network. (B) Representative western blot for pan-S6K and its T389 phosphorylated form (P-S6K) in SMC lines, and densitometry based quantification expressed as relative to control 120ls-SMCs. Average diameter of cells (C) and change in LC3B-II levels with or without bafilomycin A1 (D) in SMC lines. (E) Western blot for HIF1α (left panel) and densitometry-based quantification (right panel) expressed as relative to 121-SMC control. (F) Densitometry-based quantification of western blots for HIF1α expression in SMC lines in the presence of DMSO (vehicle) or rapamycin, expressed as relative to the DMSO condition for each cell line. Statistics are relative to 120ls-SMC (B, C, D), 121-SMC (E), and DMSO condition (F) (t-test). P values of

Techniques Used: Derivative Assay, Western Blot, Expressing, T-Test

Selective toxicity of TSC-LAM patient SMCs by dual targeting of mTORC1 and autophagy signaling. (A) Representative western blots of LC3B-I and LC3B-II levels in control (120ls-SMCs) and P6-derived SMC lines to depict LC3B-II accumulation in vehicle versus +bafilomycin A1 conditions, and the corresponding response to mTOR inhibitors torin-1 and rapamycin. (B) Basal levels of LC3B-II, based on densitometry quantification of western blots and normalized to β-actin levels, in SMC lines under vehicle, torin-1 or rapamycin treatment. Statistics are relative to vehicle. (C) % of cells positive for propidium iodide (PI) relative to vehicle for each cell line, following a 24 hr treatment with 2.5 µM chloroquine (CQ), 20 nM rapamycin, or both. Measurements were collected via flow cytometry. P values of
Figure Legend Snippet: Selective toxicity of TSC-LAM patient SMCs by dual targeting of mTORC1 and autophagy signaling. (A) Representative western blots of LC3B-I and LC3B-II levels in control (120ls-SMCs) and P6-derived SMC lines to depict LC3B-II accumulation in vehicle versus +bafilomycin A1 conditions, and the corresponding response to mTOR inhibitors torin-1 and rapamycin. (B) Basal levels of LC3B-II, based on densitometry quantification of western blots and normalized to β-actin levels, in SMC lines under vehicle, torin-1 or rapamycin treatment. Statistics are relative to vehicle. (C) % of cells positive for propidium iodide (PI) relative to vehicle for each cell line, following a 24 hr treatment with 2.5 µM chloroquine (CQ), 20 nM rapamycin, or both. Measurements were collected via flow cytometry. P values of

Techniques Used: Laser Capture Microdissection, Western Blot, Derivative Assay, Flow Cytometry, Cytometry

7) Product Images from "Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin"

Article Title: Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku278

Non-canonical regulation of EREG by TSC2 and the effect of TSC2 overexpression on mTORC2. ( A ) Western blot analysis to assess the levels of EREG, p-S6K1, S6K1, p-SGK1 and SGK1 following the treatment of cells from stable clones with 100 nM rapamycin (+) or vehicle control DMSO (−). β-actin was used as a loading control. ( B ) qRT-PCR analysis of EREG following the treatment of cells from stable clones with 100 nM rapamycin (+) or DMSO (−). RPL35A was used as a normalizing control. ns, data not significant. ( C ) Western blot analysis of cells from stable clones to assess the levels of SGK1 and p-SGK1. β-actin was used as a loading control.
Figure Legend Snippet: Non-canonical regulation of EREG by TSC2 and the effect of TSC2 overexpression on mTORC2. ( A ) Western blot analysis to assess the levels of EREG, p-S6K1, S6K1, p-SGK1 and SGK1 following the treatment of cells from stable clones with 100 nM rapamycin (+) or vehicle control DMSO (−). β-actin was used as a loading control. ( B ) qRT-PCR analysis of EREG following the treatment of cells from stable clones with 100 nM rapamycin (+) or DMSO (−). RPL35A was used as a normalizing control. ns, data not significant. ( C ) Western blot analysis of cells from stable clones to assess the levels of SGK1 and p-SGK1. β-actin was used as a loading control.

Techniques Used: Over Expression, Western Blot, Clone Assay, Quantitative RT-PCR

8) Product Images from "mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity"

Article Title: mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI73935

Efficacy of mTOR inhibition by PP242 or rapamycin in the kidney.
Figure Legend Snippet: Efficacy of mTOR inhibition by PP242 or rapamycin in the kidney.

Techniques Used: Inhibition

9) Product Images from "Preclinical evidence of the enhanced effectiveness of combined rapamycin and AICAR in reducing kidney cancer"

Article Title: Preclinical evidence of the enhanced effectiveness of combined rapamycin and AICAR in reducing kidney cancer

Journal: Molecular Oncology

doi: 10.1002/1878-0261.12370

Significant increase in number of apoptotic cells is dependent on drug concentration and time exposure in 786‐O cells. Serial concentrations of (A) rapamycin (0–100 n m ), (B) AICAR (0–10 m m ), and (C) drug combinations (0/0–2/20, 4/40 and 10/100, m m /n m ) show that increase in number of apoptotic cells is dose‐dependent using annexin V‐ FITC conjugated to PI by flow cytometry. In addition, treatment of the cells with drug combinations for 24, 48, and 72 h (D) shows that increase in number of apoptotic cells is time‐dependent. Apoptotic data were confirmed in cells by measuring apoptotic protein expression. Lysates from cells treated with rapamycin (20 n m ), AICAR (2 m m ) or rapamycin+ AICAR (20 n m /2 m m ) for 72 h were subjected to Western blot analysis to measure PARP and caspase 3 cleavages. (E) Significant increase was detected in cleavage of PARP at 85 kD a and caspase 3 at 22 and 17 kDa in 786‐O cells treated with drug combinations compared with cells treated with each drug alone for 72 h. GAPDH was used as a loading control. Data represent mean ± SEM ( n = 4). Significant difference from control tissues is indicated by * P
Figure Legend Snippet: Significant increase in number of apoptotic cells is dependent on drug concentration and time exposure in 786‐O cells. Serial concentrations of (A) rapamycin (0–100 n m ), (B) AICAR (0–10 m m ), and (C) drug combinations (0/0–2/20, 4/40 and 10/100, m m /n m ) show that increase in number of apoptotic cells is dose‐dependent using annexin V‐ FITC conjugated to PI by flow cytometry. In addition, treatment of the cells with drug combinations for 24, 48, and 72 h (D) shows that increase in number of apoptotic cells is time‐dependent. Apoptotic data were confirmed in cells by measuring apoptotic protein expression. Lysates from cells treated with rapamycin (20 n m ), AICAR (2 m m ) or rapamycin+ AICAR (20 n m /2 m m ) for 72 h were subjected to Western blot analysis to measure PARP and caspase 3 cleavages. (E) Significant increase was detected in cleavage of PARP at 85 kD a and caspase 3 at 22 and 17 kDa in 786‐O cells treated with drug combinations compared with cells treated with each drug alone for 72 h. GAPDH was used as a loading control. Data represent mean ± SEM ( n = 4). Significant difference from control tissues is indicated by * P

Techniques Used: Concentration Assay, Flow Cytometry, Cytometry, Expressing, Western Blot

Drug combinations are more effective in reducing kidney tumorigenesis. A macroscopic view of all mice kidneys was examined and tumor size was measured along three axes as cm 3 (length, width, and height). (A) Images of kidney from four groups of mice showing the tumor size in each group. (B) Data calculated from the tumor size from each group show that treatment with drug combination resulted in an 80% reduction in tumor size, treatment with rapamycin a 38% reduction, and treatment with AICAR a 36% reduction compared with control mice, further confirming the reduction of tumor size measured by bioluminescent imaging. Values represent the mean ± SEM ( n = 5). Significant difference from control mice is indicated by * P
Figure Legend Snippet: Drug combinations are more effective in reducing kidney tumorigenesis. A macroscopic view of all mice kidneys was examined and tumor size was measured along three axes as cm 3 (length, width, and height). (A) Images of kidney from four groups of mice showing the tumor size in each group. (B) Data calculated from the tumor size from each group show that treatment with drug combination resulted in an 80% reduction in tumor size, treatment with rapamycin a 38% reduction, and treatment with AICAR a 36% reduction compared with control mice, further confirming the reduction of tumor size measured by bioluminescent imaging. Values represent the mean ± SEM ( n = 5). Significant difference from control mice is indicated by * P

Techniques Used: Mouse Assay, Imaging

Drug combinations significantly decreased cell migration and cell invasion of kidney cancer cells. 786‐O cells treated with rapamycin (20 n m ), AICAR (2 m m ) or a drug combination (20 n m rapamycin/2 m m AICAR ) for 72 h were added into the upper well chamber of a 96‐well plate. (A, B) Cells treated with drug combination have a significantly low number of migrated cells and (C, D) invaded cells compared with cells treated with a single drug or control cells. The total number of migrated and invaded cells was counted using counting software and the images of migrated or invaded cells were taken using a Nikon light inverted microscope. Data represent mean ± SEM ( n = 4). Significant difference from control tissues is indicated by * P
Figure Legend Snippet: Drug combinations significantly decreased cell migration and cell invasion of kidney cancer cells. 786‐O cells treated with rapamycin (20 n m ), AICAR (2 m m ) or a drug combination (20 n m rapamycin/2 m m AICAR ) for 72 h were added into the upper well chamber of a 96‐well plate. (A, B) Cells treated with drug combination have a significantly low number of migrated cells and (C, D) invaded cells compared with cells treated with a single drug or control cells. The total number of migrated and invaded cells was counted using counting software and the images of migrated or invaded cells were taken using a Nikon light inverted microscope. Data represent mean ± SEM ( n = 4). Significant difference from control tissues is indicated by * P

Techniques Used: Migration, Software, Inverted Microscopy

Drug combination and single‐drug treatment caused no change in BW and no toxicity in treated mice. (A) No changes in BW were detected between four groups of mice during 4 weeks of treatment with single or a drug combination. (B) Renal injury was determined by measuring the urinary excretion of GST in mice following treatment with rapamycin, AICAR or combination of rapamycin+ AICAR for 4 weeks. GST activity is represented as μmol/mL/min. There were no significant changes between four groups of mice, indicating the absence of toxicity of treatment with a single drug or drug combinations. An additive effect of drug combinations resulted in significant decreases in kidney tumor size. (C) One million 786‐O cells expressing luciferase were injected in the kidney capsule of nude mice. Ten minutes prior to imaging, animals were injected with luciferin and the bioluminescent image signals were recorded using the Xenogen IVIS System. Representative images show the tumor in each of four groups. (D) Total photon radiance (p/s/cm 2 /sr) in the tumor area was measured by bioluminescent imaging and blotted to show the differences in photon flux/s between four groups. Photon data were calculated and the percentage of changes in tumor sizes between the four groups was summarized. (E) Data show that treatment with drug combinations resulted in a decreased tumor radiance of 75%, compared with 32.2% and 38% in mice treated with rapamycin and AICAR , respectively. Values represent the mean ± SEM ( n = 5). Significant difference from control mice is indicated by * P
Figure Legend Snippet: Drug combination and single‐drug treatment caused no change in BW and no toxicity in treated mice. (A) No changes in BW were detected between four groups of mice during 4 weeks of treatment with single or a drug combination. (B) Renal injury was determined by measuring the urinary excretion of GST in mice following treatment with rapamycin, AICAR or combination of rapamycin+ AICAR for 4 weeks. GST activity is represented as μmol/mL/min. There were no significant changes between four groups of mice, indicating the absence of toxicity of treatment with a single drug or drug combinations. An additive effect of drug combinations resulted in significant decreases in kidney tumor size. (C) One million 786‐O cells expressing luciferase were injected in the kidney capsule of nude mice. Ten minutes prior to imaging, animals were injected with luciferin and the bioluminescent image signals were recorded using the Xenogen IVIS System. Representative images show the tumor in each of four groups. (D) Total photon radiance (p/s/cm 2 /sr) in the tumor area was measured by bioluminescent imaging and blotted to show the differences in photon flux/s between four groups. Photon data were calculated and the percentage of changes in tumor sizes between the four groups was summarized. (E) Data show that treatment with drug combinations resulted in a decreased tumor radiance of 75%, compared with 32.2% and 38% in mice treated with rapamycin and AICAR , respectively. Values represent the mean ± SEM ( n = 5). Significant difference from control mice is indicated by * P

Techniques Used: Mouse Assay, Activity Assay, Expressing, Luciferase, Injection, Imaging

Decrease in cell proliferation by drug combinations is dependent on the drug concentration and duration of exposure in 786‐O cells. Serial concentrations of (A) rapamycin (0–100 n m ), (B) AICAR (0–10 m m ), and (C) drug combinations (0/0–2/20, 4/40 and 10/100, m m /n m ) show that the decrease in cell proliferation is dose‐dependent using the 3 H‐thymidine incorporation assay. In addition, treatment of the cells with drug combinations for 24, 48, and 72 h (D) show that the significant decrease in cell proliferation is time‐dependent. Cell proliferation data were confirmed in cells by measuring proliferative protein expression. Lysates from cells treated with rapamycin (20 n m ), AICAR (2 m m ) or rapamycin+ AICAR (20 n m /2 m m ) for 72 h were subjected to Western blot analysis to measure PCNA and cyclin D1. (E) A significant decrease in expression of PCNA and cyclin D1 was detected in cells treated with a single drug, whereas drug combinations abolished the expression of both proteins detected in cells, providing evidence of the additive effect of drug combinations in reducing cell proliferation. A combination of drugs abolished Akt survival kinase and blocked HIF ‐2α and VEGF in 786‐O cells. Cells were treated with rapamycin (20 n m ), AICAR (2 m m ) or drug combination (20 n m rapamycin/2 m m AICAR ) for 72 h. Cell lysates were subjected to Western blot analysis to measure p‐Akt, HIF ‐2α, and VEGF expression. (F, G) A significant decrease in p‐Akt, HIF ‐2α, and VEGF expression was seen in cells treated with single drug; however, the additive effect of drug combinations showed complete abolishment in HIF ‐2α and VEGF as well as in phosphorylation of Akt at Ser 473 expression compared with cells treated with single drug and control cells. GAPDH was used as a loading control. Further evidence of the effect of drug combinations on HIF ‐2α function was measured by luciferase assay. (H) The additive effect of drug combinations caused a more than 90% decrease in the promoter activity of HIF ‐2α compared with control cells using the Luciferase Reporter Assay System normalized to Renilla activity and measured by a luminometer. Data represent mean ± SEM ( n = 4). Significant difference from control tissues is indicated by * P
Figure Legend Snippet: Decrease in cell proliferation by drug combinations is dependent on the drug concentration and duration of exposure in 786‐O cells. Serial concentrations of (A) rapamycin (0–100 n m ), (B) AICAR (0–10 m m ), and (C) drug combinations (0/0–2/20, 4/40 and 10/100, m m /n m ) show that the decrease in cell proliferation is dose‐dependent using the 3 H‐thymidine incorporation assay. In addition, treatment of the cells with drug combinations for 24, 48, and 72 h (D) show that the significant decrease in cell proliferation is time‐dependent. Cell proliferation data were confirmed in cells by measuring proliferative protein expression. Lysates from cells treated with rapamycin (20 n m ), AICAR (2 m m ) or rapamycin+ AICAR (20 n m /2 m m ) for 72 h were subjected to Western blot analysis to measure PCNA and cyclin D1. (E) A significant decrease in expression of PCNA and cyclin D1 was detected in cells treated with a single drug, whereas drug combinations abolished the expression of both proteins detected in cells, providing evidence of the additive effect of drug combinations in reducing cell proliferation. A combination of drugs abolished Akt survival kinase and blocked HIF ‐2α and VEGF in 786‐O cells. Cells were treated with rapamycin (20 n m ), AICAR (2 m m ) or drug combination (20 n m rapamycin/2 m m AICAR ) for 72 h. Cell lysates were subjected to Western blot analysis to measure p‐Akt, HIF ‐2α, and VEGF expression. (F, G) A significant decrease in p‐Akt, HIF ‐2α, and VEGF expression was seen in cells treated with single drug; however, the additive effect of drug combinations showed complete abolishment in HIF ‐2α and VEGF as well as in phosphorylation of Akt at Ser 473 expression compared with cells treated with single drug and control cells. GAPDH was used as a loading control. Further evidence of the effect of drug combinations on HIF ‐2α function was measured by luciferase assay. (H) The additive effect of drug combinations caused a more than 90% decrease in the promoter activity of HIF ‐2α compared with control cells using the Luciferase Reporter Assay System normalized to Renilla activity and measured by a luminometer. Data represent mean ± SEM ( n = 4). Significant difference from control tissues is indicated by * P

Techniques Used: Concentration Assay, Thymidine Incorporation Assay, Expressing, Western Blot, Luciferase, Activity Assay, Reporter Assay

Combination of drugs reduces the binding of HIF ‐2α to the Akt promoter element. (A) EMSA analysis of DNA probe corresponding to the putative HIF ‐2α binding site in the Akt promoter was performed. Labeled probes were incubated with nuclear extracts isolated from 786‐O cells treated with rapamycin, AICAR or rapamycin+ AICAR showed significant decrease binding of HIF ‐2α to the Akt promoter. (B) Specificity of binding of HIF ‐α to the Akt promoter was confirmed by adding HIF ‐1α, HIF ‐2α or IgG antibody to the reaction mixture. (C) Further confirmation of the binding site of HIF ‐2 to Akt was obtained by incubation of the nuclear extract with mutated HIF ‐2α probe. Data confirmed that HIF ‐2α but not HIF ‐1α was a part of the DNA /protein complex, and that the mutations in the binding sequence of HIF ‐2 to Akt prevent the binding of HIF ‐2 to the DNA /protein complex. These data provide new evidence that HIF 2α is major transcription factor regulating cell survival kinase Akt.
Figure Legend Snippet: Combination of drugs reduces the binding of HIF ‐2α to the Akt promoter element. (A) EMSA analysis of DNA probe corresponding to the putative HIF ‐2α binding site in the Akt promoter was performed. Labeled probes were incubated with nuclear extracts isolated from 786‐O cells treated with rapamycin, AICAR or rapamycin+ AICAR showed significant decrease binding of HIF ‐2α to the Akt promoter. (B) Specificity of binding of HIF ‐α to the Akt promoter was confirmed by adding HIF ‐1α, HIF ‐2α or IgG antibody to the reaction mixture. (C) Further confirmation of the binding site of HIF ‐2 to Akt was obtained by incubation of the nuclear extract with mutated HIF ‐2α probe. Data confirmed that HIF ‐2α but not HIF ‐1α was a part of the DNA /protein complex, and that the mutations in the binding sequence of HIF ‐2 to Akt prevent the binding of HIF ‐2 to the DNA /protein complex. These data provide new evidence that HIF 2α is major transcription factor regulating cell survival kinase Akt.

Techniques Used: Binding Assay, Labeling, Incubation, Isolation, Sequencing

10) Product Images from "Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) regulates TOR signaling and cell growth during Drosophila development"

Article Title: Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) regulates TOR signaling and cell growth during Drosophila development

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

doi: 10.1073/pnas.1219333110

Altered TOR signaling in dPIP4K 29 larvae. ( A ) Rate of pupariation of wild-type and dPIP4K 29 larvae grown posthatching on fly food containing 2 μM rapamycin or an equal volume of vehicle control ( n = 80). ( B ) Rate of pupariation of larvae overexpressing
Figure Legend Snippet: Altered TOR signaling in dPIP4K 29 larvae. ( A ) Rate of pupariation of wild-type and dPIP4K 29 larvae grown posthatching on fly food containing 2 μM rapamycin or an equal volume of vehicle control ( n = 80). ( B ) Rate of pupariation of larvae overexpressing

Techniques Used:

11) Product Images from "Rapamycin-induced miR-30a downregulation inhibits senescence of VSMCs by targeting Beclin1"

Article Title: Rapamycin-induced miR-30a downregulation inhibits senescence of VSMCs by targeting Beclin1

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2019.4074

Rapamycin inhibits senescence in VSMCs and alleviates cell cycle arrest. Young and aging VSMCs were treated with 20 nM rapamycin for 12 h. (A) Cell senescence was measured by SA-β-gal staining. Representative images are shown. Scale bar, 200 µ m. (B) SA-β-gal-positive cell rates in the different groups. (C) Representative plots and (D) quantification of flow cytometry analysis for cell cycle phase distribution. (E) Protein expression levels of p16, p21, p53 and SA-β-gal were determined by western blotting. Representative blots are shown. (F) Quantitative analysis of indicated proteins. Results are presented as mean ± standard deviation (n=3). * P
Figure Legend Snippet: Rapamycin inhibits senescence in VSMCs and alleviates cell cycle arrest. Young and aging VSMCs were treated with 20 nM rapamycin for 12 h. (A) Cell senescence was measured by SA-β-gal staining. Representative images are shown. Scale bar, 200 µ m. (B) SA-β-gal-positive cell rates in the different groups. (C) Representative plots and (D) quantification of flow cytometry analysis for cell cycle phase distribution. (E) Protein expression levels of p16, p21, p53 and SA-β-gal were determined by western blotting. Representative blots are shown. (F) Quantitative analysis of indicated proteins. Results are presented as mean ± standard deviation (n=3). * P

Techniques Used: Staining, Flow Cytometry, Cytometry, Expressing, Western Blot, Standard Deviation

Rapamycin promotes autophagy in VSMCs by inhibiting miR-30a. VSMCs were transfected with miR-30a mimics or negative control for 48 h, and then treated with 20 nM rapamycin for 12 h. (A) Protein expression levels of LC3, Beclin1, p62, mTOR, p-mTOR, p-S6K1 and p-4EBP1 were determined by western blotting. Representative blots are shown. (B) Quantitative analysis of indicated proteins. (C) Immunofluorescence analysis for LC3B. Results are presented as mean ± standard deviation (n=3). * P
Figure Legend Snippet: Rapamycin promotes autophagy in VSMCs by inhibiting miR-30a. VSMCs were transfected with miR-30a mimics or negative control for 48 h, and then treated with 20 nM rapamycin for 12 h. (A) Protein expression levels of LC3, Beclin1, p62, mTOR, p-mTOR, p-S6K1 and p-4EBP1 were determined by western blotting. Representative blots are shown. (B) Quantitative analysis of indicated proteins. (C) Immunofluorescence analysis for LC3B. Results are presented as mean ± standard deviation (n=3). * P

Techniques Used: Transfection, Negative Control, Expressing, Western Blot, Immunofluorescence, Standard Deviation

miR-30a directly downregulates Beclin1. (A) Relative expression levels of miR-30a and (B) Beclin1 were determined by reverse transcription-quantitative polymerase chain reaction in aging VSMCs transfected with miR-30a mimics or negative control for 48 h followed by 20 nM rapamycin for 12 h. (C) Representative blots and (D) quantification of Beclin1 protein expression levels in aging VSMCs transfected with miR-30a mimics or negative control for 48 h followed by 20 nM rapamycin for 12 h. (E) The predicted binding site of miR-30a on Beclin1 3′-UTR in both the rat and human genes. A mutant binding site was constructed and the red letters indicate mutated nucleotides. (F) Relative luciferase activity was evaluated. Results are presented as mean ± standard deviation (n=3). * P
Figure Legend Snippet: miR-30a directly downregulates Beclin1. (A) Relative expression levels of miR-30a and (B) Beclin1 were determined by reverse transcription-quantitative polymerase chain reaction in aging VSMCs transfected with miR-30a mimics or negative control for 48 h followed by 20 nM rapamycin for 12 h. (C) Representative blots and (D) quantification of Beclin1 protein expression levels in aging VSMCs transfected with miR-30a mimics or negative control for 48 h followed by 20 nM rapamycin for 12 h. (E) The predicted binding site of miR-30a on Beclin1 3′-UTR in both the rat and human genes. A mutant binding site was constructed and the red letters indicate mutated nucleotides. (F) Relative luciferase activity was evaluated. Results are presented as mean ± standard deviation (n=3). * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Binding Assay, Mutagenesis, Construct, Luciferase, Activity Assay, Standard Deviation

Rapamycin alleviates senescence and cell cycle arrest in VSMCs by inhibiting miR-30a. VSMCs were transfected with miR-30a mimics or negative control for 48 h, and then treated with 20 nM rapamycin for 12 h. (A) Senescence was determined by SA-β-gal staining. Representative images are shown. Scale bar, 300 µ m. (B) SA-β-gal-positive cell rates in the different groups. (C) Representative plots and (D) quantification of flow cytometry analysis for cell cycle phase distribution. (E) Protein expression levels of p16, p21, p53 and SA-β-gal were determined by western blotting. Representative blots are shown. (F) Quantitative analysis of indicated proteins. Results are presented as mean ± standard deviation (n=3). * P
Figure Legend Snippet: Rapamycin alleviates senescence and cell cycle arrest in VSMCs by inhibiting miR-30a. VSMCs were transfected with miR-30a mimics or negative control for 48 h, and then treated with 20 nM rapamycin for 12 h. (A) Senescence was determined by SA-β-gal staining. Representative images are shown. Scale bar, 300 µ m. (B) SA-β-gal-positive cell rates in the different groups. (C) Representative plots and (D) quantification of flow cytometry analysis for cell cycle phase distribution. (E) Protein expression levels of p16, p21, p53 and SA-β-gal were determined by western blotting. Representative blots are shown. (F) Quantitative analysis of indicated proteins. Results are presented as mean ± standard deviation (n=3). * P

Techniques Used: Transfection, Negative Control, Staining, Flow Cytometry, Cytometry, Expressing, Western Blot, Standard Deviation

Rapamycin inhibits miR-30a expression and promotes autophagy in VSMCs. (A) Relative expression levels of miR-30a and (B) Beclin1 were determined by reverse transcription-quantitative polymerase chain reaction in young and aging VSMCs treated with 20 nM rapamycin for 12 h. (C) Representative blots of LC3, Beclin1, p-Beclin1, p62, mTOR, p-mTOR, p-S6K1 and p-4EBP1 protein expression levels in young and aging VSMCs treated with 20 nM rapamycin for 12 h. (D) Quantitative analysis for LC3-II/LC3-I ratio, Beclin1, p62, mTOR and p-mTOR protein expression. (E) Immunofluorescence analysis for LC3B in young and aging VSMCs treated with 20 nM rapamycin for 12 h. Results are presented as mean ± standard deviation (n=3). * P
Figure Legend Snippet: Rapamycin inhibits miR-30a expression and promotes autophagy in VSMCs. (A) Relative expression levels of miR-30a and (B) Beclin1 were determined by reverse transcription-quantitative polymerase chain reaction in young and aging VSMCs treated with 20 nM rapamycin for 12 h. (C) Representative blots of LC3, Beclin1, p-Beclin1, p62, mTOR, p-mTOR, p-S6K1 and p-4EBP1 protein expression levels in young and aging VSMCs treated with 20 nM rapamycin for 12 h. (D) Quantitative analysis for LC3-II/LC3-I ratio, Beclin1, p62, mTOR and p-mTOR protein expression. (E) Immunofluorescence analysis for LC3B in young and aging VSMCs treated with 20 nM rapamycin for 12 h. Results are presented as mean ± standard deviation (n=3). * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Standard Deviation

12) Product Images from "Ischemic postconditioning facilitates brain recovery after stroke by promoting Akt/mTOR activity in nude rats"

Article Title: Ischemic postconditioning facilitates brain recovery after stroke by promoting Akt/mTOR activity in nude rats

Journal: Journal of neurochemistry

doi: 10.1111/jnc.12342

The acute effects of rapamycin injection on protein levels of GAP43 after stroke
Figure Legend Snippet: The acute effects of rapamycin injection on protein levels of GAP43 after stroke

Techniques Used: Injection

Effects of rapamycin on protein levels of pmTOR and pS6K
Figure Legend Snippet: Effects of rapamycin on protein levels of pmTOR and pS6K

Techniques Used:

13) Product Images from "Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways"

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200605140

Ca 2+ sensor STIM1, but not the Ca 2+ channel subunit TRPC1, is required for lacritin-dependent NFATC1 translocation and mitogenesis. (A) Western blots of purified nuclei revealing that lacritin-dependent NFATC1 translocation is inhibited by extracellular EGTA and CsA and requires PKCα and STIM1, but not TRPC1. siRNA depletion of mTOR may slightly decrease NFATC1. (B) Lacritin promotes COX2 mRNA expression downstream of both NFAT and mTOR, as suggested by respective CsA and rapamycin inhibition. Similarly, COX2 expression is decreased or absent in cells depleted of NFATC1 and mTOR. (C) Cells depleted of NFATC1, mTOR, or STIM1, but not TRPC1, display little or no lacritin mitogenesis. (D) CsA and rapamycin both inhibit lacritin-dependent mitogenesis.
Figure Legend Snippet: Ca 2+ sensor STIM1, but not the Ca 2+ channel subunit TRPC1, is required for lacritin-dependent NFATC1 translocation and mitogenesis. (A) Western blots of purified nuclei revealing that lacritin-dependent NFATC1 translocation is inhibited by extracellular EGTA and CsA and requires PKCα and STIM1, but not TRPC1. siRNA depletion of mTOR may slightly decrease NFATC1. (B) Lacritin promotes COX2 mRNA expression downstream of both NFAT and mTOR, as suggested by respective CsA and rapamycin inhibition. Similarly, COX2 expression is decreased or absent in cells depleted of NFATC1 and mTOR. (C) Cells depleted of NFATC1, mTOR, or STIM1, but not TRPC1, display little or no lacritin mitogenesis. (D) CsA and rapamycin both inhibit lacritin-dependent mitogenesis.

Techniques Used: Translocation Assay, Western Blot, Purification, Expressing, Inhibition

14) Product Images from "Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions"

Article Title: Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1002/pro.2866

Kinetics of UnaG complementation assay upon addition of HBSS without FK506. A. Time‐lapse green fluorescence images of HEK293 cells expressing the UnaG fragments. B. Time‐lapse red fluorescence images of HEK293 cells. The cells were preincubated with 100 nM rapamycin, which was washed away before addition of HBSS without FK506. Numbers in images refer to time (minutes:seconds).
Figure Legend Snippet: Kinetics of UnaG complementation assay upon addition of HBSS without FK506. A. Time‐lapse green fluorescence images of HEK293 cells expressing the UnaG fragments. B. Time‐lapse red fluorescence images of HEK293 cells. The cells were preincubated with 100 nM rapamycin, which was washed away before addition of HBSS without FK506. Numbers in images refer to time (minutes:seconds).

Techniques Used: Fluorescence, Expressing

Structure‐guided design of a reversible green fluorogenic PCA. A. Structure‐guided selection of 3 split sites of UnaG. B. Schematic diagram of UnaG‐based PCA. C. Construct of Rapamycin‐inducible UnaG complementation assay. Frb and FKBP are fused to the two parts of split UnaG, separated by a T2A site. mCherry is coexpressed with a T2A site. D. Live cell imaging of the three split UnaG constructs.
Figure Legend Snippet: Structure‐guided design of a reversible green fluorogenic PCA. A. Structure‐guided selection of 3 split sites of UnaG. B. Schematic diagram of UnaG‐based PCA. C. Construct of Rapamycin‐inducible UnaG complementation assay. Frb and FKBP are fused to the two parts of split UnaG, separated by a T2A site. mCherry is coexpressed with a T2A site. D. Live cell imaging of the three split UnaG constructs.

Techniques Used: Selection, Construct, Live Cell Imaging

Kinetics of UnaG complementation assay upon formation of FKBP and Frb complex. A. Time‐lapse fluorescence images of HEK293 cells expressing the UnaG fragments. Numbers in images refer to time (minutes:seconds). B. Time‐dependent green fluorescence upon addition of rapamycin.
Figure Legend Snippet: Kinetics of UnaG complementation assay upon formation of FKBP and Frb complex. A. Time‐lapse fluorescence images of HEK293 cells expressing the UnaG fragments. Numbers in images refer to time (minutes:seconds). B. Time‐dependent green fluorescence upon addition of rapamycin.

Techniques Used: Fluorescence, Expressing

Kinetics of UnaG complementation assay upon inhibition of FKBP and Frb complex. The cells were preincubated with 100 nM rapamycin, which was washed away before addition of the FKBP and Frb inhibitor FK506, followed by time lapse imaging. A. Time‐lapse green fluorescence images of HEK293 cells expressing the UnaG fragments. B. Time‐lapse red fluorescence images of HEK293 cells. C. Time‐dependent green fluorescence normalized by mCherry upon addition of the FKBP and Frb inhibitor FK506. D. Time‐lapse green fluorescence images of HEK293 cells expressing the split Venus fragments fused to FKBP and Frb. E. Time‐lapse red fluorescence images of HEK293 cells coexpressed with the split Venus fused to FKBP and Frb. F. Time‐dependent green fluorescence normalized by mCherry upon addition of the FKBP and Frb inhibitor FK506. Numbers in images (A ‐ D) refer to time (minutes:seconds).
Figure Legend Snippet: Kinetics of UnaG complementation assay upon inhibition of FKBP and Frb complex. The cells were preincubated with 100 nM rapamycin, which was washed away before addition of the FKBP and Frb inhibitor FK506, followed by time lapse imaging. A. Time‐lapse green fluorescence images of HEK293 cells expressing the UnaG fragments. B. Time‐lapse red fluorescence images of HEK293 cells. C. Time‐dependent green fluorescence normalized by mCherry upon addition of the FKBP and Frb inhibitor FK506. D. Time‐lapse green fluorescence images of HEK293 cells expressing the split Venus fragments fused to FKBP and Frb. E. Time‐lapse red fluorescence images of HEK293 cells coexpressed with the split Venus fused to FKBP and Frb. F. Time‐dependent green fluorescence normalized by mCherry upon addition of the FKBP and Frb inhibitor FK506. Numbers in images (A ‐ D) refer to time (minutes:seconds).

Techniques Used: Inhibition, Imaging, Fluorescence, Expressing

15) Product Images from "Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFN?, Perforin, and TNF?, and due to the Elimination of Transduced Cells"

Article Title: Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFN?, Perforin, and TNF?, and due to the Elimination of Transduced Cells

Journal: Molecular Therapy

doi: 10.1038/mt.2011.243

Elimination of Ad-mediated transgene expression upon immunization is not reversed by rapamycin . C57BL/6 mice were injected with Ad-TK in the brain and 30 days later, immunized i.p. with either Ad-HPRT or saline as control. 30 days after immunization,
Figure Legend Snippet: Elimination of Ad-mediated transgene expression upon immunization is not reversed by rapamycin . C57BL/6 mice were injected with Ad-TK in the brain and 30 days later, immunized i.p. with either Ad-HPRT or saline as control. 30 days after immunization,

Techniques Used: Expressing, Mouse Assay, Injection

16) Product Images from "bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway"

Article Title: bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway

Journal: Scientific Reports

doi: 10.1038/srep09287

bFGF activates the Akt/mTOR signaling pathways and inhibits rapamycin-induced protein ubiquitination after 12 h treatment in H9C2 cells. (A) Protein expression and optical density analysis of Ub and SQSTM1/p-62 (Thr269/Ser272) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group in H9C2 cells. (B) Protein expression and the optical density analysis of p-AKT (Ser473) and p-mTOR (Ser2448) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group of H9C2 cells. * P
Figure Legend Snippet: bFGF activates the Akt/mTOR signaling pathways and inhibits rapamycin-induced protein ubiquitination after 12 h treatment in H9C2 cells. (A) Protein expression and optical density analysis of Ub and SQSTM1/p-62 (Thr269/Ser272) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group in H9C2 cells. (B) Protein expression and the optical density analysis of p-AKT (Ser473) and p-mTOR (Ser2448) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group of H9C2 cells. * P

Techniques Used: Expressing

Silencing of p62 partially attenuates the anti-apoptosis effect of bFGF, whereas shRNA against the autophagic machinery Atg7 prolongs the survival of cells co-treated with bFGF and rapamycin. (A, B) Protein expression and optical density analysis of Ub and p62 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si p62: p62 siRNA). (C, D) H9C2 cells (si co: random siRNA, si p62: p62 siRNA) were treated with 100 ng/ml rapamycin, with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells.
Figure Legend Snippet: Silencing of p62 partially attenuates the anti-apoptosis effect of bFGF, whereas shRNA against the autophagic machinery Atg7 prolongs the survival of cells co-treated with bFGF and rapamycin. (A, B) Protein expression and optical density analysis of Ub and p62 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si p62: p62 siRNA). (C, D) H9C2 cells (si co: random siRNA, si p62: p62 siRNA) were treated with 100 ng/ml rapamycin, with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells.

Techniques Used: shRNA, Expressing, Staining, Flow Cytometry, Cytometry

The protective effect of bFGF in I/R is inhibited by rapamycin. (A) The echocardiography analysis of I/R, I/R rats treated with rapamycin, bFGF and bFGF with rapamycin. The left ventricular dimension at end-diastole and end-systole (LVEDd, LVEDs), FS = 100 × (LVEDd – LVEDs)/LVEDd. * P
Figure Legend Snippet: The protective effect of bFGF in I/R is inhibited by rapamycin. (A) The echocardiography analysis of I/R, I/R rats treated with rapamycin, bFGF and bFGF with rapamycin. The left ventricular dimension at end-diastole and end-systole (LVEDd, LVEDs), FS = 100 × (LVEDd – LVEDs)/LVEDd. * P

Techniques Used:

The anti-apoptosis effects of bFGF are related to the inhibition of autophagy, which is induced by rapamycin. (A) H9C2 cells were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells. (B) A bar diagram of the apoptotic cell rate from three separate experiments. ** P
Figure Legend Snippet: The anti-apoptosis effects of bFGF are related to the inhibition of autophagy, which is induced by rapamycin. (A) H9C2 cells were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells. (B) A bar diagram of the apoptotic cell rate from three separate experiments. ** P

Techniques Used: Inhibition, Staining, Flow Cytometry, Cytometry

The cardioprotective effects of bFGF are related to the inhibition of autophagy induced by rapamycin. (A) Immunofluorescence staining results of LC3 (green), in which the nuclei are labeled with Hoechst (blue). (B, C) The protein expression of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. The optical density analysis of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. (D) H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA) were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with TUNEL. (E) Protein expression of LC3II/LC3I ATG-7, Ub, SQSTM1/p-62 (Thr269/Ser272) and cleaved-caspase-3 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA). * P
Figure Legend Snippet: The cardioprotective effects of bFGF are related to the inhibition of autophagy induced by rapamycin. (A) Immunofluorescence staining results of LC3 (green), in which the nuclei are labeled with Hoechst (blue). (B, C) The protein expression of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. The optical density analysis of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. (D) H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA) were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with TUNEL. (E) Protein expression of LC3II/LC3I ATG-7, Ub, SQSTM1/p-62 (Thr269/Ser272) and cleaved-caspase-3 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA). * P

Techniques Used: Inhibition, Immunofluorescence, Staining, Labeling, Expressing, TUNEL Assay

17) Product Images from "bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway"

Article Title: bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway

Journal: Scientific Reports

doi: 10.1038/srep09287

bFGF activates the Akt/mTOR signaling pathways and inhibits rapamycin-induced protein ubiquitination after 12 h treatment in H9C2 cells. (A) Protein expression and optical density analysis of Ub and SQSTM1/p-62 (Thr269/Ser272) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group in H9C2 cells. (B) Protein expression and the optical density analysis of p-AKT (Ser473) and p-mTOR (Ser2448) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group of H9C2 cells. * P
Figure Legend Snippet: bFGF activates the Akt/mTOR signaling pathways and inhibits rapamycin-induced protein ubiquitination after 12 h treatment in H9C2 cells. (A) Protein expression and optical density analysis of Ub and SQSTM1/p-62 (Thr269/Ser272) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group in H9C2 cells. (B) Protein expression and the optical density analysis of p-AKT (Ser473) and p-mTOR (Ser2448) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group of H9C2 cells. * P

Techniques Used: Expressing

Silencing of p62 partially attenuates the anti-apoptosis effect of bFGF, whereas shRNA against the autophagic machinery Atg7 prolongs the survival of cells co-treated with bFGF and rapamycin. (A, B) Protein expression and optical density analysis of Ub and p62 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si p62: p62 siRNA). (C, D) H9C2 cells (si co: random siRNA, si p62: p62 siRNA) were treated with 100 ng/ml rapamycin, with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells.
Figure Legend Snippet: Silencing of p62 partially attenuates the anti-apoptosis effect of bFGF, whereas shRNA against the autophagic machinery Atg7 prolongs the survival of cells co-treated with bFGF and rapamycin. (A, B) Protein expression and optical density analysis of Ub and p62 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si p62: p62 siRNA). (C, D) H9C2 cells (si co: random siRNA, si p62: p62 siRNA) were treated with 100 ng/ml rapamycin, with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells.

Techniques Used: shRNA, Expressing, Staining, Flow Cytometry, Cytometry

The protective effect of bFGF in I/R is inhibited by rapamycin. (A) The echocardiography analysis of I/R, I/R rats treated with rapamycin, bFGF and bFGF with rapamycin. The left ventricular dimension at end-diastole and end-systole (LVEDd, LVEDs), FS = 100 × (LVEDd – LVEDs)/LVEDd. * P
Figure Legend Snippet: The protective effect of bFGF in I/R is inhibited by rapamycin. (A) The echocardiography analysis of I/R, I/R rats treated with rapamycin, bFGF and bFGF with rapamycin. The left ventricular dimension at end-diastole and end-systole (LVEDd, LVEDs), FS = 100 × (LVEDd – LVEDs)/LVEDd. * P

Techniques Used:

The anti-apoptosis effects of bFGF are related to the inhibition of autophagy, which is induced by rapamycin. (A) H9C2 cells were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells. (B) A bar diagram of the apoptotic cell rate from three separate experiments. ** P
Figure Legend Snippet: The anti-apoptosis effects of bFGF are related to the inhibition of autophagy, which is induced by rapamycin. (A) H9C2 cells were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells. (B) A bar diagram of the apoptotic cell rate from three separate experiments. ** P

Techniques Used: Inhibition, Staining, Flow Cytometry, Cytometry

The cardioprotective effects of bFGF are related to the inhibition of autophagy induced by rapamycin. (A) Immunofluorescence staining results of LC3 (green), in which the nuclei are labeled with Hoechst (blue). (B, C) The protein expression of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. The optical density analysis of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. (D) H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA) were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with TUNEL. (E) Protein expression of LC3II/LC3I ATG-7, Ub, SQSTM1/p-62 (Thr269/Ser272) and cleaved-caspase-3 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA). * P
Figure Legend Snippet: The cardioprotective effects of bFGF are related to the inhibition of autophagy induced by rapamycin. (A) Immunofluorescence staining results of LC3 (green), in which the nuclei are labeled with Hoechst (blue). (B, C) The protein expression of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. The optical density analysis of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. (D) H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA) were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with TUNEL. (E) Protein expression of LC3II/LC3I ATG-7, Ub, SQSTM1/p-62 (Thr269/Ser272) and cleaved-caspase-3 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA). * P

Techniques Used: Inhibition, Immunofluorescence, Staining, Labeling, Expressing, TUNEL Assay

18) Product Images from "bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway"

Article Title: bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway

Journal: Scientific Reports

doi: 10.1038/srep09287

bFGF activates the Akt/mTOR signaling pathways and inhibits rapamycin-induced protein ubiquitination after 12 h treatment in H9C2 cells. (A) Protein expression and optical density analysis of Ub and SQSTM1/p-62 (Thr269/Ser272) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group in H9C2 cells. (B) Protein expression and the optical density analysis of p-AKT (Ser473) and p-mTOR (Ser2448) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group of H9C2 cells. * P
Figure Legend Snippet: bFGF activates the Akt/mTOR signaling pathways and inhibits rapamycin-induced protein ubiquitination after 12 h treatment in H9C2 cells. (A) Protein expression and optical density analysis of Ub and SQSTM1/p-62 (Thr269/Ser272) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group in H9C2 cells. (B) Protein expression and the optical density analysis of p-AKT (Ser473) and p-mTOR (Ser2448) of the sham, rapamycin group, and rapamycin treated with bFGF or 3-MA group of H9C2 cells. * P

Techniques Used: Expressing

Silencing of p62 partially attenuates the anti-apoptosis effect of bFGF, whereas shRNA against the autophagic machinery Atg7 prolongs the survival of cells co-treated with bFGF and rapamycin. (A, B) Protein expression and optical density analysis of Ub and p62 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si p62: p62 siRNA). (C, D) H9C2 cells (si co: random siRNA, si p62: p62 siRNA) were treated with 100 ng/ml rapamycin, with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells.
Figure Legend Snippet: Silencing of p62 partially attenuates the anti-apoptosis effect of bFGF, whereas shRNA against the autophagic machinery Atg7 prolongs the survival of cells co-treated with bFGF and rapamycin. (A, B) Protein expression and optical density analysis of Ub and p62 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si p62: p62 siRNA). (C, D) H9C2 cells (si co: random siRNA, si p62: p62 siRNA) were treated with 100 ng/ml rapamycin, with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells.

Techniques Used: shRNA, Expressing, Staining, Flow Cytometry, Cytometry

The protective effect of bFGF in I/R is inhibited by rapamycin. (A) The echocardiography analysis of I/R, I/R rats treated with rapamycin, bFGF and bFGF with rapamycin. The left ventricular dimension at end-diastole and end-systole (LVEDd, LVEDs), FS = 100 × (LVEDd – LVEDs)/LVEDd. * P
Figure Legend Snippet: The protective effect of bFGF in I/R is inhibited by rapamycin. (A) The echocardiography analysis of I/R, I/R rats treated with rapamycin, bFGF and bFGF with rapamycin. The left ventricular dimension at end-diastole and end-systole (LVEDd, LVEDs), FS = 100 × (LVEDd – LVEDs)/LVEDd. * P

Techniques Used:

The anti-apoptosis effects of bFGF are related to the inhibition of autophagy, which is induced by rapamycin. (A) H9C2 cells were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells. (B) A bar diagram of the apoptotic cell rate from three separate experiments. ** P
Figure Legend Snippet: The anti-apoptosis effects of bFGF are related to the inhibition of autophagy, which is induced by rapamycin. (A) H9C2 cells were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry. The lower right panel indicates the apoptotic cells. (B) A bar diagram of the apoptotic cell rate from three separate experiments. ** P

Techniques Used: Inhibition, Staining, Flow Cytometry, Cytometry

The cardioprotective effects of bFGF are related to the inhibition of autophagy induced by rapamycin. (A) Immunofluorescence staining results of LC3 (green), in which the nuclei are labeled with Hoechst (blue). (B, C) The protein expression of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. The optical density analysis of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. (D) H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA) were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with TUNEL. (E) Protein expression of LC3II/LC3I ATG-7, Ub, SQSTM1/p-62 (Thr269/Ser272) and cleaved-caspase-3 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA). * P
Figure Legend Snippet: The cardioprotective effects of bFGF are related to the inhibition of autophagy induced by rapamycin. (A) Immunofluorescence staining results of LC3 (green), in which the nuclei are labeled with Hoechst (blue). (B, C) The protein expression of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. The optical density analysis of LC3II/LC3I, ATG-7, ATG-5 and beclin-1 in the sham, rapa group, and rapa treated with bFGF or 3-MA. (D) H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA) were treated with 100 ng/ml rapamycin with or without 40 ng/ml bFGF. The cells were collected and stained with TUNEL. (E) Protein expression of LC3II/LC3I ATG-7, Ub, SQSTM1/p-62 (Thr269/Ser272) and cleaved-caspase-3 of the rapamycin group, and rapamycin treated with bFGF in H9C2 cells (si co: random siRNA, si ATG-7: ATG-7 siRNA). * P

Techniques Used: Inhibition, Immunofluorescence, Staining, Labeling, Expressing, TUNEL Assay

Related Articles

Diagnostic Assay:

Article Title: mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity
Article Snippet: Urine samples were sent to Stanford University Department of Comparative Medicine Diagnostic Laboratory for determination of urine parameters. .. Similar experiments were performed with rapamycin or AZD8055; 1.5 mg/kg rapamycin (Sigma-Aldrich) and 15 mg/kg AZD8055 (SelleckChem) were prepared using the same vehicle as described for the PP242 solution.

Clone Assay:

Article Title: Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin
Article Snippet: Treatment of cells with rapamycin The mTOR inhibitor, rapamycin (Sigma-Aldrich, St Louis, MO, USA) was dissolved in DMSO (Sigma-Aldrich, St Louis, MO, USA) and stored at −20°C. .. Cells from stable clones were seeded separately a day prior to the treatment in 25 cm2 flasks (6 × 106 cells/flask).

CCK-8 Assay:

Article Title: Sirolimus and metformin synergistically inhibit hepatocellular carcinoma cell proliferation and improve long-term survival in patients with HCC related to hepatitis B virus induced cirrhosis after liver transplantation
Article Snippet: Cell viability assay Cells were treated with either vehicle (control), rapamycin (Sigma, 20 nmol/L, 5μM) [ ], metformin (Enzo Life Sciences, 0, 12.5, 25, 50, 100, and 200μM), or rapamycin (Sigma) plus metformin for 12, 24, 36, and 48 h, respectively. .. Cell viability assay Cells were treated with either vehicle (control), rapamycin (Sigma, 20 nmol/L, 5μM) [ ], metformin (Enzo Life Sciences, 0, 12.5, 25, 50, 100, and 200μM), or rapamycin (Sigma) plus metformin for 12, 24, 36, and 48 h, respectively.

Cytometry:

Article Title: Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFN?, Perforin, and TNF?, and due to the Elimination of Transduced Cells
Article Snippet: Mice were euthanized 5 days after irradiation, as they cannot survive longer following lethal irradiation; spleens were kept to quantitate the levels of CD4+ and CD8+ T cells by flow cytometry and to assess the frequency of adenovirus-specific IFNγ-secreting T lymphocyte precursors by ELISPOT. .. For immunosupression by rapamycin, mice were treated with 3 mg/kg rapamycin (Sigma-Aldrich) dissolved in 2% carboxymethylcellulose (Sigma-Aldrich) every other day for 25 days.

Article Title: Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis
Article Snippet: For rapamycin and chloroquine experiments, SMCs were cultured in SMC growth media until ~60% confluent and were treated with 10 nM rapamycin (Calbiochem, #553211), 5 µM chloroquine (Sigma, #C6628), or vehicle (DMSO at 1/1000) in basal DMEM (Gibco, #11054-020) for 24 hrs. .. Cells were then harvested for flow cytometry analysis (for each sample, media- and trypsin-harvested adherent cells were combined).

Incubation:

Article Title: Sirolimus and metformin synergistically inhibit hepatocellular carcinoma cell proliferation and improve long-term survival in patients with HCC related to hepatitis B virus induced cirrhosis after liver transplantation
Article Snippet: Cell viability assay Cells were treated with either vehicle (control), rapamycin (Sigma, 20 nmol/L, 5μM) [ ], metformin (Enzo Life Sciences, 0, 12.5, 25, 50, 100, and 200μM), or rapamycin (Sigma) plus metformin for 12, 24, 36, and 48 h, respectively. .. After addition of 10 μl CCK-8 solution to each well, cells were incubated for 1.5 h, and absorbance was measured on a microplate reader (Spectramax 190, US) at 450 nm.

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways
Article Snippet: .. 72 h after transfection, mitogenesis was assessed by coaddition of 10 nM lacritin and 2 mCi/ml [3 H]TdR (GE Healthcare) for 24 h. For rapamycin and CsA inhibition, cells that had been incubated in SF media overnight were treated with 1 μM CsA (Sigma-Aldrich) for 5 h, 100 nM rapamycin (Calbiochem) for 15 min, or both; they were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml [3 H]TdR for an additional 24 h. For RT-PCR analyses, total RNAs from cells were reverse transcribed (RETROscript; Ambion) and subjected to PCR using Super Taq DNA polymerase (Ambion) for 10 min at 95°C, followed by 35 cycles for 45 s at 95°C, 45 s at 55°C, and 90 s at 72°C. ..

Article Title: Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions
Article Snippet: For snapshots [Fig. (D)], Rapamycin+ samples had been treated with 100 nM rapamycin (Calbiochem) for 3 h prior to imaging. .. Time‐lapse microscopy was performed with the aid of an environmental control unit incubation chamber (InVivo Scientific), which maintained at 37°C.

Radioactivity:

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways
Article Snippet: After washing, radioactivity was quantitated in a scintillation counter. .. 72 h after transfection, mitogenesis was assessed by coaddition of 10 nM lacritin and 2 mCi/ml [3 H]TdR (GE Healthcare) for 24 h. For rapamycin and CsA inhibition, cells that had been incubated in SF media overnight were treated with 1 μM CsA (Sigma-Aldrich) for 5 h, 100 nM rapamycin (Calbiochem) for 15 min, or both; they were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml [3 H]TdR for an additional 24 h. For RT-PCR analyses, total RNAs from cells were reverse transcribed (RETROscript; Ambion) and subjected to PCR using Super Taq DNA polymerase (Ambion) for 10 min at 95°C, followed by 35 cycles for 45 s at 95°C, 45 s at 55°C, and 90 s at 72°C.

Activity Assay:

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways
Article Snippet: 72 h after transfection, mitogenesis was assessed by coaddition of 10 nM lacritin and 2 mCi/ml [3 H]TdR (GE Healthcare) for 24 h. For rapamycin and CsA inhibition, cells that had been incubated in SF media overnight were treated with 1 μM CsA (Sigma-Aldrich) for 5 h, 100 nM rapamycin (Calbiochem) for 15 min, or both; they were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml [3 H]TdR for an additional 24 h. For RT-PCR analyses, total RNAs from cells were reverse transcribed (RETROscript; Ambion) and subjected to PCR using Super Taq DNA polymerase (Ambion) for 10 min at 95°C, followed by 35 cycles for 45 s at 95°C, 45 s at 55°C, and 90 s at 72°C. .. PLD activity was measured as previously described by .

Expressing:

Article Title: Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFN?, Perforin, and TNF?, and due to the Elimination of Transduced Cells
Article Snippet: Brains were perfused-fixed using Tyrode's and 4% paraformaldehyde and immunohistochemistry was performed using either rabbit polyclonal TK antibody for determining transgene expression or rat anti-CD4+ antibody to determine the levels of CD4+ T-cell infiltration into the brain. .. For immunosupression by rapamycin, mice were treated with 3 mg/kg rapamycin (Sigma-Aldrich) dissolved in 2% carboxymethylcellulose (Sigma-Aldrich) every other day for 25 days.

Article Title: Piecemeal Microautophagy of Nucleus in Saccharomyces cerevisiae
Article Snippet: Basal levels of PCUP1 -NVJ1- EYFP expression in YPD or SCGlu were achieved by withholding exogenous Cu2+ . .. If cells were to be starved for nitrogen (SD-N) or glucose (SC-Glu) before imaging, they were first stained with FM4-64, and then washed 5× in SD-N or SC-Glu and resuspended in SD-N or SC-Glu media for 3 h. DNA was stained immediately before imaging with 5 μM Hoechst reagent H-1398 (Molecular Probes, Eugene, OR) in SCGlu or SD-N. For rapamycin treatment, cells in SCGlu were treated for 3 h with a final concentration of 0.2 μg/ml rapamycin by using a stock solution of 20 μg/ml rapamycin (Calbiochem, San Diego, CA) in 90% ethanol/10% Triton X-100.

Modification:

Article Title: Rapamycin-induced miR-30a downregulation inhibits senescence of VSMCs by targeting Beclin1
Article Snippet: Aortic VSMCs were then cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, Merck KGaA, Darmstradt, Germany) supplemented with 10% Gibco fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 µ g/ml penicillin-streptomycin (Sigma-Aldrich; Merck KGaA) at 37°C and 5% CO2 . .. For the groups treated with rapamycin, VSMCs were treated with 20 nM rapamycin (Sigma-Aldrich; Merck KGaA) for 12 h. The untreated cells were used as controls.

Western Blot:

Article Title: Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin
Article Snippet: Treatment of cells with rapamycin The mTOR inhibitor, rapamycin (Sigma-Aldrich, St Louis, MO, USA) was dissolved in DMSO (Sigma-Aldrich, St Louis, MO, USA) and stored at −20°C. .. Post treatments, the cells were harvested and either used to prepare total protein lysates for the western blot analysis or for the isolation of total RNA for determining the level of EREG as described above.

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways
Article Snippet: PKCα depletion was confirmed by Western blotting. .. 72 h after transfection, mitogenesis was assessed by coaddition of 10 nM lacritin and 2 mCi/ml [3 H]TdR (GE Healthcare) for 24 h. For rapamycin and CsA inhibition, cells that had been incubated in SF media overnight were treated with 1 μM CsA (Sigma-Aldrich) for 5 h, 100 nM rapamycin (Calbiochem) for 15 min, or both; they were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml [3 H]TdR for an additional 24 h. For RT-PCR analyses, total RNAs from cells were reverse transcribed (RETROscript; Ambion) and subjected to PCR using Super Taq DNA polymerase (Ambion) for 10 min at 95°C, followed by 35 cycles for 45 s at 95°C, 45 s at 55°C, and 90 s at 72°C.

Article Title: Preclinical evidence of the enhanced effectiveness of combined rapamycin and AICAR in reducing kidney cancer
Article Snippet: Cells were treated with serial concentrations of AICAR (0, 2, 4, 10 mm ), rapamycin (0, 20, 40, 100 nm ) or drug combinations (0/0, 2/20, 4/40, 10/100, mm /nm , AICAR/rapamycin) for 72 h. Cells were treated with drug combinations (2 mm /20 nm , AICAR/rapamycin) for 24, 48, and 72 h. AICAR was obtained from Cayman Chemical (Ann Arbor, MI, USA) and rapamycin from Sigma‐Aldrich (St. Louis, MO, USA). .. The cells were lysed in lysis buffer and used for Western blot analysis (Simone et al ., ).

Flow Cytometry:

Article Title: Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFN?, Perforin, and TNF?, and due to the Elimination of Transduced Cells
Article Snippet: Mice were euthanized 5 days after irradiation, as they cannot survive longer following lethal irradiation; spleens were kept to quantitate the levels of CD4+ and CD8+ T cells by flow cytometry and to assess the frequency of adenovirus-specific IFNγ-secreting T lymphocyte precursors by ELISPOT. .. For immunosupression by rapamycin, mice were treated with 3 mg/kg rapamycin (Sigma-Aldrich) dissolved in 2% carboxymethylcellulose (Sigma-Aldrich) every other day for 25 days.

Article Title: Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis
Article Snippet: For rapamycin and chloroquine experiments, SMCs were cultured in SMC growth media until ~60% confluent and were treated with 10 nM rapamycin (Calbiochem, #553211), 5 µM chloroquine (Sigma, #C6628), or vehicle (DMSO at 1/1000) in basal DMEM (Gibco, #11054-020) for 24 hrs. .. Cells were then harvested for flow cytometry analysis (for each sample, media- and trypsin-harvested adherent cells were combined).

Immunohistochemistry:

Article Title: Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFN?, Perforin, and TNF?, and due to the Elimination of Transduced Cells
Article Snippet: Brains were perfused-fixed using Tyrode's and 4% paraformaldehyde and immunohistochemistry was performed using either rabbit polyclonal TK antibody for determining transgene expression or rat anti-CD4+ antibody to determine the levels of CD4+ T-cell infiltration into the brain. .. For immunosupression by rapamycin, mice were treated with 3 mg/kg rapamycin (Sigma-Aldrich) dissolved in 2% carboxymethylcellulose (Sigma-Aldrich) every other day for 25 days.

Concentration Assay:

Article Title: Ischemic postconditioning facilitates brain recovery after stroke by promoting Akt/mTOR activity in nude rats
Article Snippet: .. To study whether rapamycin, an mTOR inhibitor, abolishes the long term protection of postconditioning in nude rats, rapamycin (Calbiochem, Billerica, MA, USA) was dissolved in PBS to a final concentration of 0.1 mM. ..

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

Cell Culture:

Article Title: bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway
Article Snippet: Paragraph title: Cell Culture and Viability Assay ... Based on our previous study, the cells were treated with rapamycin (100 nM), bFGF (40 ng/ml), bFGF with rapamycin or the autophagy inhibitor 3-methyladenine (3-MA, 5 mM; Sigma-Aldrich) with rapamycin.

Article Title: Rapamycin-induced miR-30a downregulation inhibits senescence of VSMCs by targeting Beclin1
Article Snippet: Paragraph title: Cell culture and treatment ... For the groups treated with rapamycin, VSMCs were treated with 20 nM rapamycin (Sigma-Aldrich; Merck KGaA) for 12 h. The untreated cells were used as controls.

Article Title: Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis
Article Snippet: .. For rapamycin and chloroquine experiments, SMCs were cultured in SMC growth media until ~60% confluent and were treated with 10 nM rapamycin (Calbiochem, #553211), 5 µM chloroquine (Sigma, #C6628), or vehicle (DMSO at 1/1000) in basal DMEM (Gibco, #11054-020) for 24 hrs. .. Cells were then harvested for flow cytometry analysis (for each sample, media- and trypsin-harvested adherent cells were combined).

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

Article Title: Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions
Article Snippet: Paragraph title: Characterization of split‐UnaG in cultured mammalian cells ... For snapshots [Fig. (D)], Rapamycin+ samples had been treated with 100 nM rapamycin (Calbiochem) for 3 h prior to imaging.

Article Title: Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) regulates TOR signaling and cell growth during Drosophila development
Article Snippet: .. To measure the effect of rapamycin on development timing, groups of 20 newly hatched larvae from 4-h collection were placed in vials containing standard fly medium with or without 2 μM rapamycin (Sigma) and cultured at 23 °C until eclosion. .. Complete experiments and appropriate control sets have been done by acquiring images on either an Olympus FV1000 laser scanning confocal microscope, using 20× objective (UPLAPO series, N.A.

Inhibition:

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways
Article Snippet: .. 72 h after transfection, mitogenesis was assessed by coaddition of 10 nM lacritin and 2 mCi/ml [3 H]TdR (GE Healthcare) for 24 h. For rapamycin and CsA inhibition, cells that had been incubated in SF media overnight were treated with 1 μM CsA (Sigma-Aldrich) for 5 h, 100 nM rapamycin (Calbiochem) for 15 min, or both; they were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml [3 H]TdR for an additional 24 h. For RT-PCR analyses, total RNAs from cells were reverse transcribed (RETROscript; Ambion) and subjected to PCR using Super Taq DNA polymerase (Ambion) for 10 min at 95°C, followed by 35 cycles for 45 s at 95°C, 45 s at 55°C, and 90 s at 72°C. ..

Imaging:

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

Article Title: Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions
Article Snippet: .. For snapshots [Fig. (D)], Rapamycin+ samples had been treated with 100 nM rapamycin (Calbiochem) for 3 h prior to imaging. ..

Reverse Transcription Polymerase Chain Reaction:

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways
Article Snippet: .. 72 h after transfection, mitogenesis was assessed by coaddition of 10 nM lacritin and 2 mCi/ml [3 H]TdR (GE Healthcare) for 24 h. For rapamycin and CsA inhibition, cells that had been incubated in SF media overnight were treated with 1 μM CsA (Sigma-Aldrich) for 5 h, 100 nM rapamycin (Calbiochem) for 15 min, or both; they were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml [3 H]TdR for an additional 24 h. For RT-PCR analyses, total RNAs from cells were reverse transcribed (RETROscript; Ambion) and subjected to PCR using Super Taq DNA polymerase (Ambion) for 10 min at 95°C, followed by 35 cycles for 45 s at 95°C, 45 s at 55°C, and 90 s at 72°C. ..

Injection:

Article Title: mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity
Article Snippet: After 3 days of acclimatization, vehicle or PP242 solution was injected i.p. at a final dose of 30 mg/kg body weight. .. Similar experiments were performed with rapamycin or AZD8055; 1.5 mg/kg rapamycin (Sigma-Aldrich) and 15 mg/kg AZD8055 (SelleckChem) were prepared using the same vehicle as described for the PP242 solution.

Article Title: Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFN?, Perforin, and TNF?, and due to the Elimination of Transduced Cells
Article Snippet: Mice were immunized with 3 × 108 iu of Ad-HPRT (i.p.) 30 days after CNS injection. .. For immunosupression by rapamycin, mice were treated with 3 mg/kg rapamycin (Sigma-Aldrich) dissolved in 2% carboxymethylcellulose (Sigma-Aldrich) every other day for 25 days.

Affinity Precipitation:

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways
Article Snippet: 72 h after transfection, mitogenesis was assessed by coaddition of 10 nM lacritin and 2 mCi/ml [3 H]TdR (GE Healthcare) for 24 h. For rapamycin and CsA inhibition, cells that had been incubated in SF media overnight were treated with 1 μM CsA (Sigma-Aldrich) for 5 h, 100 nM rapamycin (Calbiochem) for 15 min, or both; they were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml [3 H]TdR for an additional 24 h. For RT-PCR analyses, total RNAs from cells were reverse transcribed (RETROscript; Ambion) and subjected to PCR using Super Taq DNA polymerase (Ambion) for 10 min at 95°C, followed by 35 cycles for 45 s at 95°C, 45 s at 55°C, and 90 s at 72°C. .. For immunoprecipitation experiments, cells were lacritin treated for 15 min, lysed with protease inhibitors, spun, precleared using protein A–Sepharose, and then subjected to anti-PLCγ2 affinity precipitation.

Isolation:

Article Title: Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin
Article Snippet: Treatment of cells with rapamycin The mTOR inhibitor, rapamycin (Sigma-Aldrich, St Louis, MO, USA) was dissolved in DMSO (Sigma-Aldrich, St Louis, MO, USA) and stored at −20°C. .. Post treatments, the cells were harvested and either used to prepare total protein lysates for the western blot analysis or for the isolation of total RNA for determining the level of EREG as described above.

Transfection:

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways
Article Snippet: .. 72 h after transfection, mitogenesis was assessed by coaddition of 10 nM lacritin and 2 mCi/ml [3 H]TdR (GE Healthcare) for 24 h. For rapamycin and CsA inhibition, cells that had been incubated in SF media overnight were treated with 1 μM CsA (Sigma-Aldrich) for 5 h, 100 nM rapamycin (Calbiochem) for 15 min, or both; they were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml [3 H]TdR for an additional 24 h. For RT-PCR analyses, total RNAs from cells were reverse transcribed (RETROscript; Ambion) and subjected to PCR using Super Taq DNA polymerase (Ambion) for 10 min at 95°C, followed by 35 cycles for 45 s at 95°C, 45 s at 55°C, and 90 s at 72°C. ..

Article Title: Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions
Article Snippet: HEK293T/17 cells transiently transfected with Split‐UnaG were imaged in 35 mm glass bottom dishes ∼24 h after transfection. .. For snapshots [Fig. (D)], Rapamycin+ samples had been treated with 100 nM rapamycin (Calbiochem) for 3 h prior to imaging.

IF-cells:

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

Polymerase Chain Reaction:

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways
Article Snippet: .. 72 h after transfection, mitogenesis was assessed by coaddition of 10 nM lacritin and 2 mCi/ml [3 H]TdR (GE Healthcare) for 24 h. For rapamycin and CsA inhibition, cells that had been incubated in SF media overnight were treated with 1 μM CsA (Sigma-Aldrich) for 5 h, 100 nM rapamycin (Calbiochem) for 15 min, or both; they were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml [3 H]TdR for an additional 24 h. For RT-PCR analyses, total RNAs from cells were reverse transcribed (RETROscript; Ambion) and subjected to PCR using Super Taq DNA polymerase (Ambion) for 10 min at 95°C, followed by 35 cycles for 45 s at 95°C, 45 s at 55°C, and 90 s at 72°C. ..

Staining:

Article Title: Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis
Article Snippet: For rapamycin and chloroquine experiments, SMCs were cultured in SMC growth media until ~60% confluent and were treated with 10 nM rapamycin (Calbiochem, #553211), 5 µM chloroquine (Sigma, #C6628), or vehicle (DMSO at 1/1000) in basal DMEM (Gibco, #11054-020) for 24 hrs. .. Cells were stained with propidium iodide (PI) (50 ng per 5×104 – 1×105 cells) and analyzed for PI staining via flow cytometry.

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

Mouse Assay:

Article Title: mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity
Article Snippet: WT C57BL/6 or Sgk1–/– mice (8–12 weeks old) were kept in balance cages for an acclimatization period of 3 days with free access to water and food (Picolab standard diet 20, 0.3% Na; catalog no. 5053, LabDiets). .. Similar experiments were performed with rapamycin or AZD8055; 1.5 mg/kg rapamycin (Sigma-Aldrich) and 15 mg/kg AZD8055 (SelleckChem) were prepared using the same vehicle as described for the PP242 solution.

Article Title: Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFN?, Perforin, and TNF?, and due to the Elimination of Transduced Cells
Article Snippet: .. For immunosupression by rapamycin, mice were treated with 3 mg/kg rapamycin (Sigma-Aldrich) dissolved in 2% carboxymethylcellulose (Sigma-Aldrich) every other day for 25 days. .. All animals were perfused-fixed with 4% paraformaldehyde 90 days after CNS injection and processed for immunohistochemistry as previously described.

Article Title: Comparison of rapamycin schedules in mice on high-fat diet
Article Snippet: Paragraph title: Mice ... These groups on HFD were as follows: control – untreated, R/ ip group (N = 10 initially) was treated with rapamycin (LC Laboratories, Woburn, MA) via i.p. injections 1.5 mg/kg 3 times a week every other week; R/gavage group received 1.5 mg/kg rapamune (Sirolimus, rapamycin) (Cardinal Health, Syracuse, NY, USA) as gavage 3 times a week every other week; R/ drinking group received rapamycin in drinking water; Metformin group received metformin(Sigma-Aldrich, St. Louis, MO, USA) in drinking water.

Viability Assay:

Article Title: bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway
Article Snippet: Paragraph title: Cell Culture and Viability Assay ... Based on our previous study, the cells were treated with rapamycin (100 nM), bFGF (40 ng/ml), bFGF with rapamycin or the autophagy inhibitor 3-methyladenine (3-MA, 5 mM; Sigma-Aldrich) with rapamycin.

Article Title: Sirolimus and metformin synergistically inhibit hepatocellular carcinoma cell proliferation and improve long-term survival in patients with HCC related to hepatitis B virus induced cirrhosis after liver transplantation
Article Snippet: .. Cell viability assay Cells were treated with either vehicle (control), rapamycin (Sigma, 20 nmol/L, 5μM) [ ], metformin (Enzo Life Sciences, 0, 12.5, 25, 50, 100, and 200μM), or rapamycin (Sigma) plus metformin for 12, 24, 36, and 48 h, respectively. ..

Plasmid Preparation:

Article Title: Piecemeal Microautophagy of Nucleus in Saccharomyces cerevisiae
Article Snippet: PMN structures were visualized using cNVJ1 -EYFP expressed from its native promoter in the chromosome or from a plasmid (PCUP1 -NVJ1- EYFP). .. If cells were to be starved for nitrogen (SD-N) or glucose (SC-Glu) before imaging, they were first stained with FM4-64, and then washed 5× in SD-N or SC-Glu and resuspended in SD-N or SC-Glu media for 3 h. DNA was stained immediately before imaging with 5 μM Hoechst reagent H-1398 (Molecular Probes, Eugene, OR) in SCGlu or SD-N. For rapamycin treatment, cells in SCGlu were treated for 3 h with a final concentration of 0.2 μg/ml rapamycin by using a stock solution of 20 μg/ml rapamycin (Calbiochem, San Diego, CA) in 90% ethanol/10% Triton X-100.

Irradiation:

Article Title: Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFN?, Perforin, and TNF?, and due to the Elimination of Transduced Cells
Article Snippet: Mice were euthanized 5 days after irradiation, as they cannot survive longer following lethal irradiation; spleens were kept to quantitate the levels of CD4+ and CD8+ T cells by flow cytometry and to assess the frequency of adenovirus-specific IFNγ-secreting T lymphocyte precursors by ELISPOT. .. For immunosupression by rapamycin, mice were treated with 3 mg/kg rapamycin (Sigma-Aldrich) dissolved in 2% carboxymethylcellulose (Sigma-Aldrich) every other day for 25 days.

Radio Immunoprecipitation:

Article Title: Comparison of rapamycin schedules in mice on high-fat diet
Article Snippet: .. These groups on HFD were as follows: control – untreated, R/ ip group (N = 10 initially) was treated with rapamycin (LC Laboratories, Woburn, MA) via i.p. injections 1.5 mg/kg 3 times a week every other week; R/gavage group received 1.5 mg/kg rapamune (Sirolimus, rapamycin) (Cardinal Health, Syracuse, NY, USA) as gavage 3 times a week every other week; R/ drinking group received rapamycin in drinking water; Metformin group received metformin(Sigma-Aldrich, St. Louis, MO, USA) in drinking water. ..

Time-lapse Microscopy:

Article Title: Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions
Article Snippet: For snapshots [Fig. (D)], Rapamycin+ samples had been treated with 100 nM rapamycin (Calbiochem) for 3 h prior to imaging. .. Time‐lapse microscopy was performed with the aid of an environmental control unit incubation chamber (InVivo Scientific), which maintained at 37°C.

Immunoprecipitation:

Article Title: Restricted epithelial proliferation by lacritin via PKC?-dependent NFAT and mTOR pathways
Article Snippet: 72 h after transfection, mitogenesis was assessed by coaddition of 10 nM lacritin and 2 mCi/ml [3 H]TdR (GE Healthcare) for 24 h. For rapamycin and CsA inhibition, cells that had been incubated in SF media overnight were treated with 1 μM CsA (Sigma-Aldrich) for 5 h, 100 nM rapamycin (Calbiochem) for 15 min, or both; they were then stimulated with 10 nM lacritin in the same medium with 2 mCi/ml [3 H]TdR for an additional 24 h. For RT-PCR analyses, total RNAs from cells were reverse transcribed (RETROscript; Ambion) and subjected to PCR using Super Taq DNA polymerase (Ambion) for 10 min at 95°C, followed by 35 cycles for 45 s at 95°C, 45 s at 55°C, and 90 s at 72°C. .. For immunoprecipitation experiments, cells were lacritin treated for 15 min, lysed with protease inhibitors, spun, precleared using protein A–Sepharose, and then subjected to anti-PLCγ2 affinity precipitation.

Enzyme-linked Immunospot:

Article Title: Immune-mediated Loss of Transgene Expression From Virally Transduced Brain Cells Is Irreversible, Mediated by IFN?, Perforin, and TNF?, and due to the Elimination of Transduced Cells
Article Snippet: Mice were euthanized 5 days after irradiation, as they cannot survive longer following lethal irradiation; spleens were kept to quantitate the levels of CD4+ and CD8+ T cells by flow cytometry and to assess the frequency of adenovirus-specific IFNγ-secreting T lymphocyte precursors by ELISPOT. .. For immunosupression by rapamycin, mice were treated with 3 mg/kg rapamycin (Sigma-Aldrich) dissolved in 2% carboxymethylcellulose (Sigma-Aldrich) every other day for 25 days.

Lysis:

Article Title: Preclinical evidence of the enhanced effectiveness of combined rapamycin and AICAR in reducing kidney cancer
Article Snippet: Cells were treated with serial concentrations of AICAR (0, 2, 4, 10 mm ), rapamycin (0, 20, 40, 100 nm ) or drug combinations (0/0, 2/20, 4/40, 10/100, mm /nm , AICAR/rapamycin) for 72 h. Cells were treated with drug combinations (2 mm /20 nm , AICAR/rapamycin) for 24, 48, and 72 h. AICAR was obtained from Cayman Chemical (Ann Arbor, MI, USA) and rapamycin from Sigma‐Aldrich (St. Louis, MO, USA). .. The cells were lysed in lysis buffer and used for Western blot analysis (Simone et al ., ).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • N/A
    If you like our new generation Superdex Increase and Superose Increase size exclusion chromatography SEC columns but need to purify larger volumes of up to 8 5 mL we can
      Buy from Supplier

    93
    Millipore mtorc1 inhibitor rapamycin
    Signaling pathways regulated by liraglutide. The molecular pathways shown in gray boxes illustrate our novel observations. Akt: Protein kinase B (PKB), AMPAR: α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, BDNF: brain-derived neurotrophic factor, ERK: extracellular signal-regulated kinase, GLP-1R: glucagon-like peptide 1 receptor, GluA1: AMPA receptor subunit GluR1, <t>mTORC1:</t> mammalian target of <t>rapamycin</t> complex 1, NBQX: 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, p70S6K: P70S6 kinase, PI3K: Phosphoinositide 3-kinase, PSD-95: Post Synaptic Density 95 protein, TrKBR: tropomyosin receptor kinase B receptor, 4E-BP-1: eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1.
    Mtorc1 Inhibitor Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mtorc1 inhibitor rapamycin/product/Millipore
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mtorc1 inhibitor rapamycin - by Bioz Stars, 2020-02
    93/100 stars
      Buy from Supplier

    99
    Millipore mtor inhibitor rapamycin
    S8 cells were serum-starved and pretreated with PD-98059 [MAPK/ERK kinase (MEK) inhibitor] or <t>rapamycin</t> [mammalian target of rapamycin <t>(mTOR)</t> inhibitor] for 30 min prior to PMA and/or insulin treatment. Western blots were performed to assess the phosphorylation
    Mtor Inhibitor Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mtor inhibitor rapamycin/product/Millipore
    Average 99 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    mtor inhibitor rapamycin - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    91
    Millipore inhibitors rapamycin
    Signal transduction studies of C3 protein synthesis after incubation of U373 astrocytes with HIV IIIB. U373 astrocytes were either mock treated (−) or incubated with HIV IIIB p24 (20 ng/ml, +). In addition, SQ22536 (200 μM), dibutyrl-cAMP (100 μM), forskolin or 1,9D-forskolin (10 μM), pertussis toxin (50 ng/ml), PD98059 (50 μM), <t>rapamycin</t> (50 nM), or wortmannin (500 nM) was added to the cells. Cell culture supernatants were harvested after 3 days. The amount of C3 protein was quantified by a specific ELISA. Results are the mean ± standard deviation (SD) of triplicate determinations. Statistical significance of HIV-induced C3 production with inhibitors versus HIV-induced C3 levels without inhibitors was evaluated by Student's t test. ∗∗, P
    Inhibitors Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/inhibitors rapamycin/product/Millipore
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    inhibitors rapamycin - by Bioz Stars, 2020-02
    91/100 stars
      Buy from Supplier

    Image Search Results


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

    Journal: Frontiers in Neuroscience

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

    doi: 10.3389/fnins.2018.00756

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

    Article Snippet: Additionally, these liraglutide-induced changes were blocked by the mTORC1 inhibitor rapamycin and the AMPA receptor antagonist NBQX.

    Techniques: Derivative Assay, Binding Assay

    S8 cells were serum-starved and pretreated with PD-98059 [MAPK/ERK kinase (MEK) inhibitor] or rapamycin [mammalian target of rapamycin (mTOR) inhibitor] for 30 min prior to PMA and/or insulin treatment. Western blots were performed to assess the phosphorylation

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Insulin signaling in retinal neurons is regulated within cholesterol-enriched membrane microdomains

    doi: 10.1152/ajpendo.00641.2010

    Figure Lengend Snippet: S8 cells were serum-starved and pretreated with PD-98059 [MAPK/ERK kinase (MEK) inhibitor] or rapamycin [mammalian target of rapamycin (mTOR) inhibitor] for 30 min prior to PMA and/or insulin treatment. Western blots were performed to assess the phosphorylation

    Article Snippet: The PKC inhibitor bisindolylmaleimide-I (BimI) and the mTOR inhibitor rapamycin were from EMD Biosciences (San Diego, CA).

    Techniques: Western Blot

    Signal transduction studies of C3 protein synthesis after incubation of U373 astrocytes with HIV IIIB. U373 astrocytes were either mock treated (−) or incubated with HIV IIIB p24 (20 ng/ml, +). In addition, SQ22536 (200 μM), dibutyrl-cAMP (100 μM), forskolin or 1,9D-forskolin (10 μM), pertussis toxin (50 ng/ml), PD98059 (50 μM), rapamycin (50 nM), or wortmannin (500 nM) was added to the cells. Cell culture supernatants were harvested after 3 days. The amount of C3 protein was quantified by a specific ELISA. Results are the mean ± standard deviation (SD) of triplicate determinations. Statistical significance of HIV-induced C3 production with inhibitors versus HIV-induced C3 levels without inhibitors was evaluated by Student's t test. ∗∗, P

    Journal: Journal of Virology

    Article Title: Mechanism of Human Immunodeficiency Virus-Induced Complement Expression in Astrocytes and Neurons

    doi: 10.1128/JVI.76.7.3179-3188.2002

    Figure Lengend Snippet: Signal transduction studies of C3 protein synthesis after incubation of U373 astrocytes with HIV IIIB. U373 astrocytes were either mock treated (−) or incubated with HIV IIIB p24 (20 ng/ml, +). In addition, SQ22536 (200 μM), dibutyrl-cAMP (100 μM), forskolin or 1,9D-forskolin (10 μM), pertussis toxin (50 ng/ml), PD98059 (50 μM), rapamycin (50 nM), or wortmannin (500 nM) was added to the cells. Cell culture supernatants were harvested after 3 days. The amount of C3 protein was quantified by a specific ELISA. Results are the mean ± standard deviation (SD) of triplicate determinations. Statistical significance of HIV-induced C3 production with inhibitors versus HIV-induced C3 levels without inhibitors was evaluated by Student's t test. ∗∗, P

    Article Snippet: The inhibitors rapamycin, wortmannin, SQ22536 [9-(tetrahydro-2′-furyl)adenine] and PD98059 were from Calbiochem (San Diego, Calif.).

    Techniques: Transduction, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation