Embryos were treated with 10 μm rapamycin (Sangon Biotech, Shanghai, China) in PTU egg water from 5 to 7 dpf and replaced rapamycin solution every 24 h. The control group was treated with 0.2% DMSO. To induce rbm15 overexpression from Tg(hsp70l:rbm15-Flag)cq97, larvae were placed in egg water and then incubated in a 38.5 °C water bath for 30 min once a day from 5 to 7 dpf.
Rapamycin
Manufactured by Sangon
Sourced in China
Rapamycin is a macrolide compound produced by the bacterium Streptomyces hygroscopicus. It functions as an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway, a crucial regulator of cell growth, proliferation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Anti phospho mtor ser2448, by Cell Signaling Technology (2 mentions)
Cyto id autophagy detection kit, by Enzo Life Sciences (2 mentions)
Chloroquine, by Merck Group (2 mentions)
Anti sqstm1, by Cell Signaling Technology (2 mentions)
Anti mtor, by Cell Signaling Technology (2 mentions)
Anti phospho p70 s6 kinase, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
Rat il 34 recombinant protein, by Sangon (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Lipopolysaccharides (lps), by Cell Signaling Technology (1 mentions)
Lipopolysaccharides (lps), by Sangon (1 mentions)
Anti mouse igg secondary antibody, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Beyotime (1 mentions)
Lysotracker dnd 99, by Thermo Fisher Scientific (1 mentions)
Anti 4e bp1, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
14 protocols using rapamycin
1
Rapamycin and rbm15 Overexpression Assay
2
Kupffer Cell Activation and Regulation
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KCs were isolated from LEW rat liver according to the method described previously, 35 and cultured in Roswell Park Memorial Institute 1640 containing 10% fetal bovine serum (FBS) as well as 1% streptomycin and penicillin. KCs were divided into the following groups: Control group (KCs cultured in normal media), lipopolysaccharide (LPS) group (KCs were treated with LPS (Cell Signaling Technology, USA) for 12 h), IL-34 group (KCs were treated with rat IL-34 recombinant protein (Cloud Clone corporation, USA) for 12 h after treatment with LPS), LY294002 group (KCs were pretreated with LY294002 (Cell Signaling Technology, USA) 2 h before treatment with rat IL-34 recombinant protein and LPS), Rapamycin group (KCs were pretreated with Rapamycin (Sangon Biotech, China) 8 h before treatment with rat IL-34 recombinant protein and LPS).
3
Nectin-4-MMAE Autophagy Modulation and Apoptosis Assay
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Nectin-4-MMAE is a gift from Mabwell Bioscience CO., LTD. Shanghai China. Autophagy modulators: LY294002 (Medchemexpress, HY-10108, Shanghai China); Chloroquine (Sigma-Aldrich, C6628, Darmstadt Germany); Rapamycin (Sangon Biotech, A606203, Shanghai China). Cell viability assay: MTT (Beyotime, C0009S, Shanghai, China). ADC labeling: AlexaFlour 488 protein label kit (ThermoFisher, A10235, MA US). Cell apoptosis assay: cell apoptosis detection kit (Meilunbio MA0220-1, Dalian China). Autophagy assay: Cyto-ID autophagy detection kit (Enzo Life Sciences, ENZO-51031-K200, NY US); Lyso-Tracker-DND 99 (Invitrogen, L7528, CA US). Antibodies: Primary antibody: anti-GAPDH, #2118; anti-LC3 I/II, #3868; anti-SQSTM1, #8025; anti-caspase 3, #9662; anti-PARP, #9542; anti-phospho-mTOR (Ser2448), #2971; anti-mTOR, #2983; anti-phospho-AKT (Ser473), #4060; anti-phospho-p70s6 Kinase (Ser371), #9208; anti-p70s6 Kinase, #2708; anti-phospho-4EBP1 (Thr45), #2971; anti-4EBP1, #9644, anti-PI3 Kinase, #4257; anti-phospho-PI3 Kinase, #4228 (Cell Signaling Technology, MA US). Anti-PDK-1, AF7707 (Beyotime Biotechnology, Shanghai, China). Anti-PTEN, A19104 (Abclonal, Wuhan China). Secondary antibody: HRP-conjugated anti-rabbit secondary antibody, #7074; anti-mouse IgG secondary antibody, #7076 (Cell Signaling Technology, MA US).
4
Bovine Mammary Epithelial Cell Culture
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The procedures of BMEC culture were described previously (Wang et al., 2018 (link)). The mammary gland tissues were acquired from 3 midlactation dairy cows at a local abattoir. The use of animal tissues was approved by the Institutional Animal Use Committee of Zhejiang University. Briefly, the minced tissues collected from bovine mammary gland were digested in trypsin (0.25%, Amesco, Solon, OH, USA) at 37 °C for 30 min and subsequently in collagenase I (Amesco) and collagenase II (Amesco) for 4 h at 37 °C. The digested tissue homogenate was filtrated and centrifuged at 300 g or 5 min, the cells were cultivated in plastic dishes containing DMEM/F12 with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), transferrin (Sigma, St. Louis, MO, USA), insulin (Sigma), prolactin (5 mg/mL, Sigma), hydrocortisone (1 mg/mL, Sigma) and epithelial growth factor (10 ng/mL, Sigma). After the cells were starved for 12 h and treated with rapamycin (100 ng/mL, Sangon, Shanghai, China) for 12 h, they were then harvested with RIPA cell lysate buffer (Beyotime, Jiangsu, China) or used to study peptide uptake. BMEC used in the study were between passages 5 and 8.
5
Regulation of Autophagy and Signaling
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If not otherwise indicated, all chemicals were from Sigma. MG132 (10 μM; Beyotime Biotechnology ) and CQ (100 μM) exposure was for 4 hr. Rapamycin (200 nM; Sangon Biotech), AICAR (500 μM; Beyotime Biotechnology ), MHY (5 μM) and dorsomorphin (5 μM) were added to the medium for 12 hr. pAC5.1-flag and pAC5.1-HA vectors were kindly provided by Xiaohang Yang (Zhejiang University). AKHR-HA was made by cloning the cDNA of AKHR into a pAC5.1-HA vector using SalI and NotI. The cDNA of GFP-ATG8 was from Chao Tong (Zhejiang University) and cloned into a pAC5.1-HA vector using EcoRI and BamHI to generate HA-GFP-ATG8.
The following antibodies were used: rabbit polyclonal antibodies to p-AKT, p-AMPK, HA, Flag, GFP and mouse monoclonal antibody to HA (Cell Signaling Technology); mouse monoclonal antibody to actin (sigma); rabbit polyclonal antibody to AKH (biorbyt); rabbit polyclonal antibody to Dilp2 was kindly provided by Zhefeng Gong (Zhejiang University).
The following antibodies were used: rabbit polyclonal antibodies to p-AKT, p-AMPK, HA, Flag, GFP and mouse monoclonal antibody to HA (Cell Signaling Technology); mouse monoclonal antibody to actin (sigma); rabbit polyclonal antibody to AKH (biorbyt); rabbit polyclonal antibody to Dilp2 was kindly provided by Zhefeng Gong (Zhejiang University).
6
Autophagy Regulation by BAG3 and CRYAB
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The cDNA of BAG3 and CRYAB were amplified using RNA extracted from HEK293 cell, followed by RT-PCR and subcloned into pLenti vector (OBIO, H139).
H2O2 (SinoPharm Chemical Reagent Co., Ltd., China. Cat. #7722-84-1). Rapamycin (Sangon Biotech, China. Cat. #A606203) and chloroquine (Sangon Biotech. Cat. #A506569) were used. The following antibodies were used: anti-LC3 (MBL, Cat. #PM036), anti-β-actin (Sigma, Cat. #A5441), anti-RPS6KB/p70S6 (Cell Signaling, Cat. #2708), anti-phospho-RPS6KB/p70S6 (Cell Signaling, Cat. #9206), anti-mTOR (Cell Signaling, Cat. #2972), anti-phospho-mTOR (Sigma, Cat. #SAB4504043), anti-SQSTM1/p62 (MBL, Cat. #PM045), anti-GAPDH (SAB, Cat. #1336), anti-CRYAB (Santa Cruz Biotech, Cat. #sc-137,143), anti-HSPB8 (Abcam, Cat. #ab4149), anti-HSC70 (Santa Cruz Biotech, Cat. #sc-7298), anti-BAG3 (Abcam, Cat. #ab47124), anti-ubiquitin (DAKO, Cat. #z0458), anti-α-synuclein (Cell Signaling, Cat. #2628), anti-GFP (Santa Cruz Biotech, Cat. #sc-9996).
H2O2 (SinoPharm Chemical Reagent Co., Ltd., China. Cat. #7722-84-1). Rapamycin (Sangon Biotech, China. Cat. #A606203) and chloroquine (Sangon Biotech. Cat. #A506569) were used. The following antibodies were used: anti-LC3 (MBL, Cat. #PM036), anti-β-actin (Sigma, Cat. #A5441), anti-RPS6KB/p70S6 (Cell Signaling, Cat. #2708), anti-phospho-RPS6KB/p70S6 (Cell Signaling, Cat. #9206), anti-mTOR (Cell Signaling, Cat. #2972), anti-phospho-mTOR (Sigma, Cat. #SAB4504043), anti-SQSTM1/p62 (MBL, Cat. #PM045), anti-GAPDH (SAB, Cat. #1336), anti-CRYAB (Santa Cruz Biotech, Cat. #sc-137,143), anti-HSPB8 (Abcam, Cat. #ab4149), anti-HSC70 (Santa Cruz Biotech, Cat. #sc-7298), anti-BAG3 (Abcam, Cat. #ab47124), anti-ubiquitin (DAKO, Cat. #z0458), anti-α-synuclein (Cell Signaling, Cat. #2628), anti-GFP (Santa Cruz Biotech, Cat. #sc-9996).
7
Zebrafish Intestinal Digestive Function
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2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) (5 mg/mL, Sigma) was used to label the zebrafish intestines at 7 dpf. Embryos were treated with DCFH-DA in 0.3× Danieau buffer for 2 h. PED6 (D23739, Thermo Fisher) and EnzChek (E6639, Thermo Fisher) were used to test the digestive ability of the intestinal proteins and lipids. Embryos at 7 dpf were treated with 3 μg/mL PED6 or 20 μg/mL EnzChek in a 0.3× Danieau buffer for 3 h. rapamycin was used to inhibit the mTORC1 pathway. Embryos were exposed to 400 and 800 nM rapamycin (Sangon Biotech, China) in a 0.3× Danieau buffer from 10 hours postfertilization (hpf) to 3 dpf. L-Leu and rheb mRNA were used to elevate the mTORC1 pathway. Embryos were injected with L-Leu (500 nM, Sigma) at 30 hpf or rheb mRNA (100 pg and 150 pg) at 1-cell stage, then harvested at 3 dpf.
8
Pharmacological Modulation of PI3K/AKT/mTOR Pathway
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The PI3K inhibitor LY294002 was obtained from Beyotime Institute of Biotechnology (Haimen, China). The lysosomal inhibitor chloroquine (CQ) and autophagy inducer trehalose were obtained from Sigma (St Louis, MO). Antioxidants N-acetyl cysteine (NAC), lipoic acid (LA), and tocopherol were purchased from Beyotime Institute of Biotechnology (Haimen, China). Bafilomycin A1 and Rapamycin were obtained from Sangon Biotech (Shanghai, China). Cyto-ID Green dye kit was purchased from ENZO Life Science (Farmingdale, NY, USA). MitoSox Red mitochondrial superoxide indicator was obtained from Life Technologies (Eugene, Oregon, USA). MEK 1/2 inhibitor U0126 was supplied by Cell Signaling Technology (Danvers, MA, USA). The antibodies used were as follows: anti-LC3B, anti-SQSTEM1/p62, anti-phospho-AKT (Ser473), anti-phospho-MTOR (Ser2448), anti-p70 S6 Kinase Phospho (pS371), anti-phospho-p44/42 MAPK (Erk 1/2) (Thr202/Tyr204), anti-tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA); the second antibodies horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit immunoglobulin G (IgG) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). All cell culture reagents were purchased from Gibco (Carlsbad, CA, USA).
9
HepG2 Cytokine and Rapamycin Effects
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The human hepatoblastoma cell line HepG2 was obtained from the American Type Culture Collection. The experiments were performed in a serum-free medium containing Dulbecco’s modified Eagle medium-high glucose, 0.2% bovine serum albumin (BSA), 0.04 mmol/L palmitate. The cells were subjected to 10 ng/mL rapamycin (Sangon Biotech, Shanghai, China) or inflammatory cytokines by loading 25 ng/mL tumour necrosis factor alpha (TNF-α, PeproTech, Rocky Hill, NJ, USA) or 20 ng/mL interleukin-6 (IL-6, Sinobio, Shanghai, China) for 24 hours.
10
Exploring DSS-Mediated Autophagy Regulation
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The NRK-52E cell line was purchased from Shanghai Enzyme Research Biotechnology Co., LTD. (Shanghai, China), in a DMEM high sugar culture medium (Gibco, NY, USA) containing 10% fetal bovine serum (FBS; Every Green, Hangzhou, China) and 100 U/ml penicillin-streptomycin (Gibco, NY, USA). Cells were incubated at 37°C and 5% CO2. Group intervention: The blank control group was cultured in a serum-free DMEM medium, and the model group was stimulated with 5 ng/ml TGF-β1 (PeproTech, NJ, USA). The treatment group was incubated with DSS and the positive drug control group was incubated with 10 nM rapamycin (Sangon Biotech, Shanghai, China) for 48 h on the basis of the model group. In order to further study the therapeutic effect of DSS, the cells were divided into the following groups: The blank control group was cultured in a serum-free DMEM medium, the autophagy-inhibited group was stimulated with 10 mM 3-MA (MCE, Shanghai, China), and the treatment group was incubated with 10 mM 3-MA + 0.8 mg/ml DSS for 24 hours.
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