TMZ (No. PHR1437, Sigma) was dissolved in dimethyl sulfoxide (DMSO) at a 100 mM stock concentration and stored at -20 °C. Autophagy inhibitor 3-MA (No. M9281, Sigma) and LY294002 (No. S1105, Selleck Chemicals) were dissolved to 10 mM and 100 μM as stock solutions. CY-09 (No. S5774, Selleck Chemicals) and FPS-ZM1 (No. S8185, Selleck Chemicals) were at 5 mM and 100 μM as stock concentrations. Recombinant human HMGB1 (rhHMGB1, No.1690-HMB-050) was purchased from R&D Systems and recombinant mouse HMGB1 (rmHMGB1, Cat. 50913-M01H) was from Sino Biological.
Autophagy inhibitor 3 ma
Manufactured by Merck Group
Sourced in United States
Autophagy inhibitor 3-MA is a laboratory compound used to inhibit the process of autophagy, which is the degradation and recycling of cellular components within the body. It functions by blocking the formation of autophagosomes, which are the vesicles responsible for carrying cellular material to the lysosome for degradation.
Automatically generated - may contain errors
Lab products found in correlation
Cy 09, by Selleck Chemicals (1 mentions)
Fps zm1, by Selleck Chemicals (1 mentions)
Ly294002, by Selleck Chemicals (1 mentions)
Z vad fmk, by Santa Cruz Biotechnology (1 mentions)
Embelin, by Santa Cruz Biotechnology (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Bt549, by American Type Culture Collection (1 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Mda mb 435, by American Type Culture Collection (1 mentions)
Ldh cytotoxicity colorimetric assay kit 2, by Abcam (1 mentions)
Lc3b 2775, by Cell Signaling Technology (1 mentions)
Secondary goat anti mouse 554002, by BD (1 mentions)
Anti rabbit 554021, by BD (1 mentions)
Versadoc mp 5000 system, by Bio-Rad (1 mentions)
β catenin sc 59737, by Santa Cruz Biotechnology (1 mentions)
β actin a5441, by Merck Group (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
8 protocols using autophagy inhibitor 3 ma
1
Preparation of Autophagy and HMGB1 Modulators
2
Induction of Autophagy in Breast Cell Lines
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Human breast cancer cell lines MCF-7, T47D, SKBR3, BT549, MDA-MB-231, MDA-MB-435S and a human breast non-tumorigenic cell line MCF-10A were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were maintained in a humidified incubator at 37°C and 5% CO2. Cells were treated with Earle's balanced salt solution (EBSS, Sigma) to activate starvation-induced autophagy [27 (link)]. Apoptosis inhibitor Z-VAD-FMK (Santa Cruz), autophagy inhibitor 3-MA (Sigma) and XIAP inhibitor Embelin (Santa Cruz) were treated when necessary.
3
Rapamycin-Induced Autophagy and Apoptosis
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Rapamycin was purchased from MedChemExpress LLC (NJ 08540, USA). MTT, protease and phosphatase inhibitors were from Sigma-Aldrich (Oakville, Ontario, Canada). LDH-Cytotoxicity Colorimetric Assay Kit II was purchased from BioVision (Milpitas, California, USA), while Annexin V-FITC/PI Kit was from BD Bioscience. (Mississauga, ON, Canada). Autophagy Assay, Red (Cat. #9156), Intracellular Total ROS Activity Assay (Cat. #9144) and Intracellular GSH Assay (Cat. #9137) were purchased from ImmunoChemistry Technologies (Davis, CA, USA). Autophagy Inhibitor, 3-MA (Cat. #189490) and N-acetylcysteine (NAC) were from Sigma. ECL system was acquired from EMD Millipore (Billerica, MA, USA). The primary antibodies as procaspase 3 (sc-56046), procaspase 9 (sc-17784), NF-κB (sc-8008) and β-catenin (sc-59737) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), E-cadherin (8834), pERK1/2 (4370), ERK1/2 (4695), pp38 (4631), p38 (9212), cleaved caspase-3 (9664S), cleaved caspase-9 (20750S) were from Cell Signaling Technology (Danvers, MA, USA), LC3B (2775) and p62 (39,749) were all from Cell Signaling Technology (Danvers, MA, USA)and β-actin (A5441) was from Sigma-Aldrich (Oakville, ON, Canada). The secondary goat anti-mouse (554002) and anti-rabbit (554021) were from BD Pharmingen (Mississauga, ON, Canada). VersaDoc™ MP 5000 system was from Bio-Rad (Mississauga, ON, Canada).
4
Modulating Autophagy and ER Stress in Cell Apoptosis
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Melatonin, ERS inhibitor 4PBA, autophagy inhibitor 3MA and Hoechst 33342 stain were obtained from Sigma-Aldrich (Merck KGaA). An Annexin V-FITC/PI Apoptosis Detection kit was obtained from Nanjing KeyGen Biotech Co., Ltd. Primary antibodies against microtubule-associated protein 1 light chain 3β (LC3; 1:1,000; cat. no. ab128025), glucose-regulated protein, 78 kDa (GRP78; 1:1,000; cat. no. ab21685), septin7 (1:1,000; cat. no. ab186021) were obtained from Abcam. Primary antibodies against poly (ADP-ribose) polymerase 1 (PARP-1; 1:1,000; cat. no. 9532), C/EBP homologous protein (CHOP; 1:1,000; cat. no. 5554), β-actin (1:1,000; cat. no. 4970) were obtained from Cell Signaling Technology, Inc., as were fluorescent anti-rabbit secondary antibodies (1:500; cat. no. 4414).
5
Autophagy Marker Antibody and Inhibitor Protocol
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Antibodies against LC3 (Cat No. 12741), ATG5 (Cat No. 12994) and cathepsin D (Cat No. 2284) were purchased from Cell Signaling Technology (CST) (MA, USA). Anti-SQSTM1/p62 (ab101266) and anti-cathepsin L (ab103574) were purchased from Abcam PLC (Abcam, Cambridge, UK). Anti-β-actin (Cat No. A00702) was obtained from GenScript Biotech Corporation. Monoclonal anti-PPV capsid protein antibody was produced by 3C9 cell clones that were obtained from the American Type Culture Collection (Cat No. ATCC CRL-1745). Polyclonal anti-NS1 antibody was prepared by our laboratory, and was acquired from rabbits immunized with purified truncated NS1 protein expressed by pET32a vector in Escherichia coli (E. coli). Autophagy flux inhibitor Bafilomycin A1 (Cat No. 54645) was purchased from Cell Signaling Technology (CST) (MA, USA). Autophagy inhibitor 3MA (Cat No. 5142-23-4) was purchased from Sigma-Aldrich (USA).
6
Autophagy Inhibition and Quantification
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Autophagy inhibitor 3-MA (Sigma-Aldrich Chemical Company, St Louis, MO, USA) was diluted using sterile PBS. The final concentration of stock solution was adjusted to 40 mM, and the stock solution was sub-packed at 20°C for further usage. MDC powder (Sigma-Aldrich Chemical Company, St Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) to obtain a final concentration of 0.1 M and then stored at -20°C. The macrophages were seeded into six-well plates, treated with 3-MA and cultured in brand-new culture medium containing 50 μm of MDC at 37°C in 5% CO 2 for 10 min. The solutions were centrifuged at 1000 rpm and resuspended for 10 min. The cells were applied to slide glass with a cover glass, observed under an inverted fluorescence microscope OLYMPUS IX71 (Olympus Corp., Tokyo, Japan), activated by ultraviolet light, and imaged.
7
ROS Levels Modulation by CONPs
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Intracellular ROS levels were determined using a ROS detection assay kit (Beyotime Biotechnology, Shanghai, China). T24 and 5637 cells were treated with CONPs at a concentration of 0.625, 1.25, or 2.5 mg/ml for 24 h. In inhibitory studies, the cells were incubated with CONPs alongside 5 mM of the ROS inhibitor N-acetylcysteine (NAC) (Beyotime Biotechnology) or 10 mM of 3-MA autophagy inhibitor (Sigma-Aldrich) for 2 h. The cells were then harvested, washed three times with PBS, and treated with 10 mM dihydrodichlorofluorescein diacetate (DCF-DA) in serum-free McCoy’s 5 A medium at 37 °C for 30 min. Then, the cells were washed 3 times with serum-free medium, after which DCF fluorescence was measured by using a MACS QuantifyTM flow cytometer (Miltenyi Biotec, Germany).
8
Evaluating Microcystin-LR Cytotoxicity
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Microcystin-LR (MC-LR) (purity ≧ 95%, by HPLC) was purchased from Express Technology Co., Ltd (Beijing, China). RPMI 1640 culture medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA), while 4-Phenyl butyric acid (4-PBA) and 3-MA autophagy inhibitor were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Cell Counting Kit-8 was purchased from Dojindo Lab (Kumamoto, Japan). Reactive oxygen species assay kit and Annexin V-FITC apoptosis detection kit were purchased from Beyotime Biotechnology Company (Nanjing, China). All other reagents were of analytical grade.
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