Age- and sex-matched wild type (WT, Harlan Sprague Dawley) and PCK rats (Harlan Sprague Dawley background), WT and Pkd2WS25/- mice and Pkhd1del2/del2 mice (all, C57BL/6 background) were housed at the 12 hours light/dark cycle, maintained on a standard diet and water ad lib. After anesthesia (pentobarbital, 50 mg/kg) livers were removed, fixed and paraffin-embedded. The protocol was approved by the Mayo Institutional Animal Care and Use Committee. Normal (NRC) and PCK rat cholangiocytes, normal human (NHC) and ADPKD cholangiocytes were maintained as we previously described.10 (link) NRC and PCK rat cholangiocytes were stable transfected with LC3-GFP construct (Invitrogen). ADPKD cholangiocytes were transfected with ATG7 or control siRNAs (Invitrogen). Bafilomycin A1 and hydroxychloroquine (HCQ) were purchased from Sigma Aldrich.
Hydroxychloroquine hcq
Manufactured by Merck Group
Sourced in United States
Hydroxychloroquine (HCQ) is a pharmaceutical compound used in a variety of laboratory and research applications. It is a synthetic derivative of the antimalarial drug chloroquine. HCQ is commonly used as a research tool in experimental studies, with applications in fields such as immunology, cell biology, and drug screening.
Automatically generated - may contain errors
Lab products found in correlation
3 methyladenine 3 ma, by Merck Group (3 mentions)
Bafilomycin a1 bafa1, by Merck Group (2 mentions)
Lc3 gfp construct, by Thermo Fisher Scientific (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
P nf κb p65, by Wanlei (1 mentions)
Cy3 goat anti rabbit igg h l, by ABclonal (1 mentions)
Nf κb p65, by Abmart (1 mentions)
Atf 4, by Wanlei (1 mentions)
Fitc goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
P iκb, by Abmart (1 mentions)
Hrp goat anti rabbit igg, by ABclonal (1 mentions)
P62 sqstm1, by Proteintech (1 mentions)
19 protocols using hydroxychloroquine hcq
1
Autophagy in Polycystic Kidney Disease
2
Tzb Modulation of Autophagy and Apoptosis
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Tzb was provided by Ningbo No. 2 Hospital (Zhejiang, China), solubilized in water (stock solution at 21 mg/ml), stored at 4°C and used within 1 month. Dimethylsulfoxide (DMSO), 3-methyladenine (3MA), MTT, crystal violet, hydroxychloroquine (HCQ) and bafilomycin A1 (BafA1) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Everolimus was provided by the China State Institute of Pharmaceutical Industry (Shanghai, China). RPMI-1640 medium, 10 U/ml penicillin-streptomycin (P/S), 0.25% trypsin, fetal bovine serum (FBS) and bovine serum albumin (BSA) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Cell lysis buffer, polyvinylidene difluoride (PVDF) membranes, and Tween-20 were purchased from Weiao Inc. (Shanghai, China). Glutaraldehyde, Epon 812, DDSA, NMA and DMP-30 were purchased from Sinopharm Inc. (Beijing, China).
3
Chalcone, Autophagy, and Cell Signaling
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(E)2’-hydroxychalcone (2’-HC; purity ≥ 98%) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Hydroxychloroquine (HCQ; purity ≥ 98%), 3-Methyladenine (3-MA; purity ≥ 98%), and N-acetylcysteine (NAC; purity ≥ 99%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin (Rap; purity ≥ 98%) was purchased from Aladdin (Shanghai, China).
The primary antibodies used were as follows: β-actin, Bax, Bcl-2, PARP, cleaved-PARP p25, caspase-9, caspase-3, LC3B, Beclin1, and p-IκB. They were purchased from ABclonal (Wuhan, China). p62/SQSTM1 was obtained from Proteintech (Wuhan, China). ERK, JNK, p-JNK, p38, p-p38, NF-κBp65, IκB, p-IκB, p-eIF2α, and MMP9 were obtained from Abmart (Shanghai, China). P-ERK, p-NF-κBp65, ATF-4, and CHOP were purchased from Wanleibio (Shenyang, China). HRP Goat Anti-Rabbit IgG (Abclonal), Alexa Flour 594-Goat Anti-Rabbit IgG (Abbox, Jiangsu, China), Cy3 Goat Anti-Rabbit IgG (H + L) (Abclonal), and FITC Goat Anti-Rabbit IgG (Servicebio, Wuhan, China) were used as secondary antibodies for a Western blot or immunofluorescence.
The primary antibodies used were as follows: β-actin, Bax, Bcl-2, PARP, cleaved-PARP p25, caspase-9, caspase-3, LC3B, Beclin1, and p-IκB. They were purchased from ABclonal (Wuhan, China). p62/SQSTM1 was obtained from Proteintech (Wuhan, China). ERK, JNK, p-JNK, p38, p-p38, NF-κBp65, IκB, p-IκB, p-eIF2α, and MMP9 were obtained from Abmart (Shanghai, China). P-ERK, p-NF-κBp65, ATF-4, and CHOP were purchased from Wanleibio (Shenyang, China). HRP Goat Anti-Rabbit IgG (Abclonal), Alexa Flour 594-Goat Anti-Rabbit IgG (Abbox, Jiangsu, China), Cy3 Goat Anti-Rabbit IgG (H + L) (Abclonal), and FITC Goat Anti-Rabbit IgG (Servicebio, Wuhan, China) were used as secondary antibodies for a Western blot or immunofluorescence.
4
Apoptosis Regulation by BCL-2 Inhibitors
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The following anti-human antibodies were used: mouse anti-SQSTM1/p62 (sc-28359) and rabbit (sc-130656) or mouse (sc-47778) anti-Actin, from Santa Cruz (Santa Cruz, CA, USA), Mouse anti-LC3 (5F10, 0231-100) from Nanotools (Munich, Germany), P62, and anti-BCL-2 (Santa Cruz, CA, USA), anti-TOM20 and anti-VMP1 were obtained from cell signalling (Leiden, the Netherlands), Hydroxychloroquine (HCQ), was obtained from Sigma-Aldrich. Venetoclax/ABT-199 (BCL-2 inhibitor, Selleckchem Munich, Germany), S63845 (MCL-1 inhibitor) was obtained from APExBIO (Boston, MA, USA). The pan caspase inhibitor ZVAD-FMK was obtained from (Enzo Life Sciences, Bruxelles, Belgium).
5
Antiviral Compound T-1105 Evaluation
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The antiviral compound T-1105 was provided by the School of Pharmacy and Pharmaceutical Sciences of the Cardiff University, UK. T-1105 is a direct nucleoside (purine) analogue and the defluorinated analogue of favipiravir (T-705). The compound was provided as a solid powder and was dissolved in DMSO to create a 10 mM solution.
Other antiviral substances used as controls were ribavirin (RBV), and hydroxychloroquine (HCQ) (both from Sigma-Aldrich). RBV and HCQ were dissolved in purified water to create stock solutions of 100 mM and 10 mM, respectively. For further dilutions DMEM LG was used.
Other antiviral substances used as controls were ribavirin (RBV), and hydroxychloroquine (HCQ) (both from Sigma-Aldrich). RBV and HCQ were dissolved in purified water to create stock solutions of 100 mM and 10 mM, respectively. For further dilutions DMEM LG was used.
6
Glioblastoma Stem Cell Cytotoxicity Assay
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GSCs were mechanically dissociated and plated at a density of 10 × 104 six-well microtiter plates. After 16 h, GSCs were treated for 72 h and 96 h.
The following chemicals and drugs were used: 450 μM Temozolomide (TMZ, Cayman Chemical Inc., Ann Arbor, MI), 10 μM z-VAD-FMK (Enzo Life Sciences, Rome, Italy), 5 μM CA074 (Chemicon International, Inc), 100 mM Trehalose (TRE, Sigma-Aldrich), 5 μM Quinacrine (QN, Sigma-Aldrich), 30 μM hydroxychloroquine (HCQ, Sigma-Aldrich), 10 μM calpain inhibitor I, 100 μM Necrostatin-1 (Enzo Life Sciences) 20 μM pepstatin A (Sigma-Aldrich), 100 μM deferoxamine (DFO, Sigma-Aldrich), and 20 μM ferrostatin 1 (Sigma-Aldrich). Compounds were dissolved in DMSO or in serum-free medium. Samples treated with vehicle alone were considered as control.
The following chemicals and drugs were used: 450 μM Temozolomide (TMZ, Cayman Chemical Inc., Ann Arbor, MI), 10 μM z-VAD-FMK (Enzo Life Sciences, Rome, Italy), 5 μM CA074 (Chemicon International, Inc), 100 mM Trehalose (TRE, Sigma-Aldrich), 5 μM Quinacrine (QN, Sigma-Aldrich), 30 μM hydroxychloroquine (HCQ, Sigma-Aldrich), 10 μM calpain inhibitor I, 100 μM Necrostatin-1 (Enzo Life Sciences) 20 μM pepstatin A (Sigma-Aldrich), 100 μM deferoxamine (DFO, Sigma-Aldrich), and 20 μM ferrostatin 1 (Sigma-Aldrich). Compounds were dissolved in DMSO or in serum-free medium. Samples treated with vehicle alone were considered as control.
7
Investigating Autophagy and Apoptosis in Cells
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The antibodies for microtubule-associated light chain 3 (LC3) (cat. no. 12741s) and cleaved-caspase-3 (cat. no. 9664s) were purchased from Cell Signaling Technology, Inc. p62 (cat. no. Ab109012), Ki67 (cat. no. Ab16667), and caspase-3 (cat. no. Ab32351) antibodies were obtained from Abcam. Anti-CD31 (cat. no. AF6191) was obtained from Affinity. Hydroxychloroquine (HCQ) was purchased from Sigma-Aldrich (Merck KGaA); Annexin V-fluorescein isothiocyanate (FITC) Assay kit was from BD Biosciences, Cell Counting Kit-8 (CCK-8, cat. no. CK04) was purchased from Dojindo Molecular Technologies, Inc., and TRIzol® (cat. no. 9109) was purchased from Thermo Fisher Scientific, Inc.
8
Cell Line Maintenance and Drug Treatments
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Huh-7.5, SK-Hep1, and HepG2 cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Carlsbad, CA), and supplemented with 2 mM L-glutamine, sodium pyruvate, nonessential amino acids, 100U/mL penicillin, 100mg/mL streptomycin and 10% fetal bovine serum (FBS). Cells were grown at 37°C in a 5% CO2 atmosphere within a humidified incubator with regular medium change at 3-day intervals. Hydroxychloroquine (HCQ) and Doxycycline was purchased from Sigma-Aldrich (St Louis, MO). Torin-1 was purchased from Cell Chem (Houston, TX). Antibodies specific for p62, Hsc70, BiP, p53 and Betaactin (Cell signaling, MA), an antibody to LAMP-2A was purchased from Abcam Ltd (USA); an antibody to glypican-3 was purchased from Biocare.
9
Neuroblastoma Cell Line Experiments
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The neuroblastoma cell line SH-SY5Y was purchased from DSMZ (Braunschweig, Germany), while SK-N-BE(2) and IMR-32 cells were obtained from ATCC (Manassas, VA). The cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 1% antibiotics, and glutamine (Gibco). The cells were then exposed to PON (Sigma-Aldrich), CQ (Sigma-Aldrich), or a combination of PON and CQ (COMBO) for the indicated times and at the specified doses. The genetic background of the cell lines is summarized in Supplementary Table S1 . In vivo studies were done with the less toxic analog hydroxychloroquine (HCQ; Sigma-Aldrich) [27 (link)]. Torin 1 (Sigma-Aldrich) was dissolved in DMSO before use, and the cell cultures were regularly tested for the presence of mycoplasmas by PCR. Human cell line authentication was done at BMR Genomics S.r.l. (Padova, Italy).
10
Inhibiting TLR9-Mediated Cellular Response
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Cells were treated with inhibitors for 1 h prior to the addition of ox-mtDNA. Inhibitors used: our developed TLR9-IgG4 chimera at 50 ng/ul, and corresponding isotype; 0.5 uM IRAK 1/4 inhibitor (Caymen 17540, CAS 509093-47-4), 1 uM ODN 4048-F (TLR9 antagonist) and ODN 2395 control (Invivogen, San Diego, CA, USA), 30 uM Hydroxychloroquine (HCQ, Sigma-Aldrich, St. Louis, MO, USA) to accumulate autophagosomes for imaging and 10 uM for CFAs, 20 ug/mL IRS95424, 1 µM RU.521 (cGAS inhibitor, Aobious, Gloucester, MA, USA).
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