The present study used NCI-H460 as a human large cell lung carcinoma, cell line, 293 cells known as the Human Embryonic Kidney 293 cells and HepG2 as a liver cancer cell line. Cells were obtained from American Type Cell Culture (ATCC, Manassas, VA) and kindly provided by Professor Longping Wen from University of Science and Technology of China. NCI-H460 cells were cultured in RPMI 1640 (SH30809.01, Hyclone) medium, and 293 and HepG2 cells were cultured in or Dulbecco's Modified Eagle medium (Hyclone; GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (FBS; Biological Industries) at 37°C in the presence of 5% CO2. Cells were treated with 50 µM chloroquine (CQ; Sigma-Aldrich; Merck KGaA) or 100 mM trehalose (Tre; autophagy inducer; Sigma-Aldrich; Merck KGaA) for 24 h (21 (link)). Sufentanil was used at the concentration of 1 nM and incubated with cells for 24 h (22 (link)). Sufentanil was purchased from Yichang Hmanwell Pharmaceutical Co., Ltd.
Chloroquine cq
Manufactured by Merck Group
Sourced in United States, China, Germany, Sao Tome and Principe, United Kingdom
Chloroquine (CQ) is a laboratory chemical product manufactured by Merck Group. It serves as a core compound for various research and analytical applications. Chloroquine is a white, crystalline powder with a molecular formula of C₁₈H₂₆ClN₃. Its primary function is as a reference standard and intermediate in chemical synthesis processes. Further details on intended use or applications are not provided to maintain an unbiased and factual approach.
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Lab products found in correlation
3 methyladenine 3 ma, by Merck Group (47 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (34 mentions)
Mg132, by Merck Group (18 mentions)
Rapamycin, by Merck Group (17 mentions)
Cycloheximide (chx), by Merck Group (17 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (17 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (16 mentions)
Bafilomycin a1 bafa1, by Merck Group (15 mentions)
Beclin 1, by Cell Signaling Technology (12 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (12 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (11 mentions)
β actin, by Cell Signaling Technology (9 mentions)
Cleaved caspase 3, by Cell Signaling Technology (9 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (8 mentions)
P mtor, by Cell Signaling Technology (7 mentions)
Mg132, by Selleck Chemicals (7 mentions)
3 methyladenine 3 ma, by Selleck Chemicals (7 mentions)
351 protocols using chloroquine cq
1
Cell Line Responses to Autophagy and Sufentanil
2
Autophagy Induction and Flux Measurement
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RAW264.7 cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium containing 4 mM l -glutamine, 4500 mg/l glucose, 1 mM sodium pyruvate, and 1500 mg/l sodium bicarbonate (all from Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). Cells were maintained at 37 °C in a humidified incubator with 5% CO2. For induction of autophagy, cells were treated with 50 nM RAPA for 30 min. The autophagic flux was measured by treating cells with 50 μM chloroquine (CQ, Sigma-Aldrich, USA), an inhibitor of lysosomal degradation, for 8 h [12 (link)].
3
Modulating Autophagy in Spinal Cord Injury
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C57BL/6 mice were randomly allocated into the Sham group, 1dpi group (1 day after SCI), 3dpi group (3 days after SCI) and 7dpi group (7 days after SCI); other mice were divided into SCI+3-MA, SCI+shRNA-Klf2 group, SCI+shRNA-Con group, SCI+LV-Klf2 group, SCI+LV-Con group and SCI+LV-Klf2+CQ group. Mice from the SCI+3-MA group and SCI+LV-Klf2+CQ group received daily intraperitoneal injections of 3-Methyladenine (3-MA, Sigma-Aldrich St. Louis, MO, USA, 15 mg/kg) and chloroquine (CQ, Sigma-Aldrich St. Louis, MO, USA, 50 mg/kg) for 3 days after SCI. The SCI contusion model was constructed according to the previous work 9 (link). Briefly, after anesthesia with 0.3% sodium pentobarbital solution (0.2 ml/10g, ip), mice underwent a laminectomy at vertebral level T9-T10 to expose the dorsal cord surface without disrupting the dura. Subsequently, a 10g weight was dropped from 2.0 cm onto the exposed dorsal surface of the spinal cord to induce a moderate SCI model. Afterward, muscle and skin were sutured in layers. After the operation, the mice received daily manual urinary bladder emptying until the bladder reflex was re-established. The sham group mice were treated with anesthesia and a laminectomy but without SCI.
4
Evaluation of CpG-ODN 7909 and Chloroquine
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CpG-ODN 7909 (5′-TCGTCGTTTTGTCGTTTTGT CGTT-3′-) was selected according to the published reports and purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, People’s Republic of China).23 (link),25 (link),26 (link) Chloroquine (CQ), a TLR9 inhibitor, was obtained from Sigma-Aldrich Co. (St Louis, MO, USA). CpG-ODN 7909 and CQ were separately resuspended in phosphate buffer saline to 1 mg/mL stock solutions and maintained at −20°C until use.
5
Autophagy Modulation in Steroidogenesis
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Cell culture medium, Dulbecco’s Modified Eagle Medium: F12 (DMEM/F12), and phosphate buffer solution (PBS) were purchased from Hyclone (USA). Fetal bovine serum was obtained from BI (Biological Industries, Israel), and TP was purchased from Beijing ChengZhiKewWei Biological Engineering (Beijing, China). Chloroquine (CQ) (cas: 5063-05), dimethyl sulfoxide (DMSO) (cas: 67-68-5), and monodansyl-cadaverine (MDC) were purchased from Sigma-Aldrich (USA). β-Actin (TA-09) and β-tubulin (TA-10) antibodies were purchased from Zhongshanjinqiao; anti-LC3B monoclonal antibody (#2775), anti-P62 monoclonal antibody (#8025), anti-mammalian rapamycin target protein (mTOR) monoclonal antibody (#2983), anti-p-mTOR monoclonal antibody (#5536) , anti-StAR monoclonal antibody (#8449), anti-p70s6k monoclonal antibody(#2708), anti-p-p70s6k monoclonal antibody (#9234) , anti-4E-BP1 monoclonal antibody (#9644) , and anti-p-4E-BP1 antibody (#2855) were purchased from Cell Signaling Technology (USA). The cell-counting kit (CCK)-8 and Annexin V-FITC/propidium iodide (PI) apoptosis assay kits were obtained from Kaiji Biological Technology (Nanjing, China).
6
Autophagy Modulation: Evaluating Molecular Mechanisms
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Sodium fluoride, thiazolyl blue tetrazolium bromide (MTT), chloroquine (CQ) (autophagy inhibitor), dimethyl sulfoxide (DMSO), rapamycin (autophagy activator) and 3-MA (autophagy inhibitor) were purchased from Sigma-Aldrich. The SIRT1 activator SRT1720 and inhibitor Ex- 527 were purchased from Selleck Chemicals. rapamycin and CQ were dissolved in PBS, 3-MA, SRT1720 and Ex-527 were dissolved in DMSO to provide a working stock solution. The DMSO concentration was maintained at 0.1% in all cell cultures, and it did not exert any detectable effect on cell growth nor cell death. Anti-LC3, anti-cleaved-caspase-3 and anti-FoxO1 antibodies were purchased commercially (Cell signal technology, CA, USA). Anti-Ac-FoxO1, HRP-conjugated goat anti-mouse and goat anti-rabbit antibodies were purchased from Santa Cruz Biotech Company. Anti-Bnip3 and anti-Rab7 were purchased from Abclonel Company.
7
Monitoring Autophagy Flux in HASMCs
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The autophagic flux was dynamically observed in HASMCs with mCherry‐GFP‐LC3B overexpression as described previously.14 To further evaluate the effect of METTL3 on autophagic flux, we further knocked down or overexpressed METTL3 in mCherry‐GFP‐LC3B‐overexpressing HASMCs. Chloroquine (CQ) (20 μM; C6628; Sigma‐Aldrich), a lysosomotropic agent, was used to inhibit the content degradation of autolysosome. After treated with indicated stimulus, the HASMCs were fixed with 4% paraformaldehyde in PBS for 10 min. The fluorescence images were acquired by using a fluorescence microscope. Yellow and red colour indicate autophagosomes or autolysosomes, respectively.
8
Investigating Cellular Mechanisms in Stress Response
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Pharmacological agents used: angiotensin II (Ang II), chloroquine (CQ), Xanthine oxidase (XO), and rapamycin (Rapa) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Rhodamine phalloidin was acquired from Cytoskeleton (Denver, CO, United States). Mito-TEMPO and 4-PBA were obtained from Sigma-Aldrich (Sigma, St. Louis, MO, United States). Antibodies against LC3A/B, ATG5, p62, mTOR, p-mTOR, ATG5, ATG7, Beclin 1, PERK, XBP1, CHOP, β-actin, and GAPDH were purchased from Cell Signaling Technology (Boston, MA, United States). GRP78, GSK3β, p-GSK3β (Ser9), β-TrcP, and NRF2 were purchased from Abcam (Cambridge, MA, United States). NOX2, NOX4, and HO-1 were provided by Proteintech (Wuhan, China).
9
Inducible C9orf72 Transgene Model in HeLa Cells
10
Regulation of Pancreatic Cancer Cell Autophagy
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Pancreatic cancer Panc1 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA), the cells were maintained in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 50 μg/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Thermo Fisher Scientific) in a 5% CO2 humidified atmosphere at 37°C.
Chloroquine (CQ), a lysosomal inhibitor, was purchased from the Sigma Aldrich (St. Louis, MO, USA). 3-methyladenine (3-MA), a PI3K inhibitor, which can also specific inhibit autophagy, was obtained from MedChemExpress (Monmouth Junction, NJ, USA). The cells were treated with CQ at 40 μM for 2 h, or 3-MA for 24 h, according to the previous report (17 (link)). MK-2206 or SCH772984 (Selleck Company, Houston, TX, USA), specific inhibitors of the Akt or ERK1/2 signaling pathways, respectively, were added to the tissue culture medium. The final concentrations were 5 μM (MK-2206) or 10 μM (SCH772984) for treatment of Panc1 cells. Untreated cells were used as a control.
Chloroquine (CQ), a lysosomal inhibitor, was purchased from the Sigma Aldrich (St. Louis, MO, USA). 3-methyladenine (3-MA), a PI3K inhibitor, which can also specific inhibit autophagy, was obtained from MedChemExpress (Monmouth Junction, NJ, USA). The cells were treated with CQ at 40 μM for 2 h, or 3-MA for 24 h, according to the previous report (17 (link)). MK-2206 or SCH772984 (Selleck Company, Houston, TX, USA), specific inhibitors of the Akt or ERK1/2 signaling pathways, respectively, were added to the tissue culture medium. The final concentrations were 5 μM (MK-2206) or 10 μM (SCH772984) for treatment of Panc1 cells. Untreated cells were used as a control.
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