The radioimmunoprecipitation assay (RIPA) lysis buffer was purchased from Solarbio (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone Laboratories, Inc. (HyClone, Logan, UT, USA). Penicillin and streptomycin were purchased from Gibco BRL (Gibco, Carlsbad, CA, USA). All other chemicals and reagents were of analytical grade. BAF, rapamycin (RAPA), and CQ were purchased from MedChem Express (MCE, Monmouth Junction, NJ, USA). The OEA used in the cellular study was purchased from Sigma-Aldrich (St. Louis, MO, USA). The OEA (purity ≥ 98%) used in the animal study was synthesized in our laboratory as previously described [18 (link)].
Rapamycin rapa
Manufactured by MedChemExpress
Sourced in United States
Rapamycin (Rapa) is a macrolide compound isolated from the bacterium Streptomyces hygroscopicus. It functions as an immunosuppressant and has been widely used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Chloroquine cq, by MedChemExpress (3 mentions)
3 methyladenine 3 ma, by MedChemExpress (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Dulbecco s modified eagle s medium dmem, by Cytiva (1 mentions)
Radioimmunoprecipitation assay ripa lysis buffer, by Solarbio (1 mentions)
Ab13248, by Abcam (1 mentions)
Ab129068, by Abcam (1 mentions)
Ab133303, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Angiotensin 2 ang 2, by Merck Group (1 mentions)
Ab32124, by Abcam (1 mentions)
Anti lc3a b, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Beyotime (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Acridine orange, by Solarbio (1 mentions)
Anti rabbit igg sa00001 2, by Proteintech (1 mentions)
Pepstatin a, by MedChemExpress (1 mentions)
8 protocols using rapamycin rapa
1
Experimental Protocols for Cellular and Animal Studies
2
Autophagy and Oxidative Stress Regulation
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ICS II (purity ≥ 98%), rapamycin (Rapa) and chloroquine (CQ) were purchased from MedChemExpress CO. (Monmouth Junction) and solubilized in 0.1% dimethyl sulphoxide (DMSO; Sigma, USA). Angiotensin II (Ang II) was purchased from Sigma‐Aldrich (St. Louis). Rabbit anti‐Nox2/gp91phox monoclonal antibody (Abcam, ab129068), rabbit anti‐NADPH oxidase 4 antibody (Abcam, ab133303), mouse anti‐haeme oxygenase 1 antibody (Abcam, ab13248), rabbit anti‐Bcl‐2 antibody (Abcam, ab32124) and rabbit anti‐Bax antibody (Abcam, ab32503) were purchased from Abcam. Rabbit anti‐LC3 I/II antibody (#4108), rabbit anti‐Atg5 antibody (#9980), rabbit anti‐Atg7 antibody (#8558), rabbit anti‐Beclin 1 antibody (#3495), rabbit anti‐p62 antibody (#23214), rabbit anti‐ACC antibody (#3662), rabbit anti‐SIRT3 antibody (#2627), rabbit anti‐p‐ACC antibody (#11818), rabbit anti‐AMPK antibody (#2532), rabbit anti‐p‐AMPK antibody (#50081), rabbit anti‐cleaved caspase 3 antibody (#9664) and rabbit anti‐GAPDH antibody were purchased from CST. Rabbit anti‐OXCT1 antibody (#12175‐1‐AP) and rabbit anti‐MCT1 antibody (#20139‐1‐AP) were purchased from Proteintech.
3
Chloroquine and Rapamycin Autophagy Modulation
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Chloroquine (CQ) was purchased from Sigma-Aldrich (St. Louis, MI, USA). Rapamycin (Rapa) was purchased from MedChemExpress, (South Brunswick, NJ, USA). Acridine orange (AO) was purchased from Solarbio (Beijing, China). EG was purchased from Macklin (Shanghai, China).
The antibodies used in this study were anti-Fragilis (Huabio, Hangzhou, China), anti-LC3A/B (Cell Signaling Technology, Danvers, MA, USA), anti-cathepsin B polyclonal antibody (Wanleibio, Shenyang, China), anti-BVDV E2-specific mouse monoclonal antibody (VMRD, Pullman, WA, USA). Additionally, anti-LAMP1 (21997-1-AP), anti-SQSTM1/p62 (18420-1-AP), anti-GAPDH (60004-1-AP), and anti-Tubulin (10068-1-AP) were purchased from ProteinTech Group Inc. (Rosemont, IL, USA). Secondary antibodies in this study included Alexa Fluor 488-Conjugated Goat Anti-Rabbit IgG (Beyotime, Shanghai, China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (SA00001-1), and anti-rabbit IgG (SA00001-2) were purchased from ProteinTech Group Inc. (Rosemont, IL, USA).
The antibodies used in this study were anti-Fragilis (Huabio, Hangzhou, China), anti-LC3A/B (Cell Signaling Technology, Danvers, MA, USA), anti-cathepsin B polyclonal antibody (Wanleibio, Shenyang, China), anti-BVDV E2-specific mouse monoclonal antibody (VMRD, Pullman, WA, USA). Additionally, anti-LAMP1 (21997-1-AP), anti-SQSTM1/p62 (18420-1-AP), anti-GAPDH (60004-1-AP), and anti-Tubulin (10068-1-AP) were purchased from ProteinTech Group Inc. (Rosemont, IL, USA). Secondary antibodies in this study included Alexa Fluor 488-Conjugated Goat Anti-Rabbit IgG (Beyotime, Shanghai, China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (SA00001-1), and anti-rabbit IgG (SA00001-2) were purchased from ProteinTech Group Inc. (Rosemont, IL, USA).
4
Autophagy Regulation by Chemical Inhibitors
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TSN (purity > 98%, MB6571) was purchased from Meilunbio Company (Dalian, China). Chloroquine (CQ, purity 99.50%, HY-17589A), rapamycin (Rapa, purity 99.81%, HY-10219), Bafilomycin A1 (Baf, purity > 99.0%, HY-100558), E64D (purity 99.55%, HY-100229), and pepstatin A (purity 98.28%, HY-P0018) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Anti-LC3B (18725-1-AP), anti-P62 (18420-1-AP), anti-LAMP2 (66201-1-Ig), and anti-GAPDH (60004-1-Ig) primary antibodies were purchased from Proteintech (Rosemont, IL, USA). Anti-LAMP1 (#15665), anti-CTSB (#31718), anti-TFEB (#37785), anti-p-TFEB(Ser211) (#37681), and anti-Histone H3(#4499) primary antibodies were purchased from Cell Signaling Technology (Billerica, CA, USA).
5
Modulation of Cellular Pathways in Prostate Cells
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In order to deactivate ERK1/2, prostate cells were pretreated with 10 μM MEK1/2 inhibitor U0126 (MedChemExpress, China) for 24 h prior to plasmid transfection. And, prostate cells were pretreated with RSL3 (MedChemExpress, China) at 0.25 μM, 0.5 μM, 1 μM, 2 μM or 4 μM for 24 h to activate ferroptosis, isosilybin B (MedChemExpress, China) at 20 μM, 40 μM, 60 μM, 80 μM or 100 μM for 24 h to activate apoptosis, Rapamycin (Rapa, MedChemExpress, China) at 0.5 nM, 1 nM, 2 nM, 4 nM or 8 nM for 24 h to activate autophagy, Chloroquine (CQ, MedChemExpress, China) at 10 μM, 20 μM, 30 μM, 40 μM or 50 μM for 24 h to inhibit autophagy, and GSK621 (MedChemExpress, China) at 5 μM, 10 μM, 20 μM, 30 μM or 40 μM for 24 h to activate AMPK. Meanwhile, equivalent amount of DMSO without drugs were added to the cells served as a control.
6
Molecular Mechanisms of Autophagy Regulation
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EDA (purity = 99.59%), 3‐Methyladenine (3‐MA, purity = 99.83%) and Rapamycin (Rapa, purity = 99.77%) were purchased from MedChem Express (New Jersey, US). Primary antibodies against transforming growth factor‐β1 (TGF‐β1), vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP9) were purchased from Wanleibio (Shenyang, China). Heme oxygenase‐1 (HO‐1), NAD(P)H quinone oxidoreductase 1 (NQO1) and β‐actin antibodies were purchased from ABclonal Technology (Wuhan, China). LC3, Beclin1, p62 and Atg5 antibodies were purchased from Affinity Biosciences (OH, US). Cleaved‐Caspase3, Bcl‐2, Bax, p‐PI3K, PI3K, p‐AKT, AKT, p‐mTOR and mTOR antibodies were purchased from Cell Signalling Technology (Danvers, MA). RPMI 1640 medium was purchased from Gibco (Grand Island, NY) and fetal bovine serum was purchased from Animal Blood Ware (Shanghai, China).
7
Ferroptosis Mechanism Evaluation Protocol
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Trypan blue solution (Prod. No. T8154), erastin (Selleck Chemicals, USA), ferrostatin-1 (Fer-1), ZVAD-FMK, 3-methyladenine (3-MA), and rapamycin (Rapa) were purchased from MedChemExpress (MCE, USA), and phosphatase inhibitor cocktails 2 and 3 (Prod. No. P5726 & P0044) were from Sigma-Aldrich (St. Louis, MO, USA). A protease inhibitor cocktail (ref no. 11836 153 001) was from Roche Diagnostics (Basel, Switzerland). Primary antibodies are as follows: AdipoR1 (ab126611), transferrin (ab8241), and GPX4 (125066) from Abcam (Cambridge, MA, USA); xCT (#12691S), CD71 (#13113S), and Nrf2 (#12721S) from Cell Signaling Technology (Danvers, MA USA); GAPDH from Proteintech (Rosemont, IL); actin (A3853) from Sigma; and AdipoR1 (SC-518030) from SANTA. Secondary antibodies are as follows: goat anti-rabbit IgG (H+L)-HRP conjugate (Cat. No. 170-6515) and goat anti-mouse IgG (H+L)-HRP conjugate (Cat. No. 170-6516) obtained from Bio-Rad Laboratories (Mississauga, ON, Canada). siRNA, targeting human xCT, and the corresponding control, siRNA NC, were purchased from GenePharma (Shanghai, China).
8
Exploring Ovarian Cancer Cell Lines
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The human ovarian cancer cell lines A2780 and OVCAR3 were gifted by Dr. Cheng Hailing (Cancer Institute, Dalian Medical University, China). The cell lines were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum, penicillin(100 U/mL)and streptomycin (100 µg/mL) in a humidi ed incubator with 5% CO2 at 37℃. All cells were used within eight passages. Olaparib was purchased from Chemexpress (China), and apatinib obtained from Hengrui Medicine Co. Ltd (Jiangsu, China). Ferrostatin-1 (Fer-1), ML385, Omaveloxolone, RITA were obtained from Abmole (USA). 3-Methyladenine (3-MA) and rapamycin (Rapa) were purchased from Medchem Express(USA).
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