The A549 cell line, which is derived from a human alveolar cell carcinoma, has many properties of human alveolar epithelial cells and was utilized because it is the most widely used in vitro model of type II pulmonary alveolar epithelial cells [38 (link)]. The cells were obtained from the American Type Culture Collection (ATCC® CCL-185TM) and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% glutamine in a humidified atmosphere with 5% CO2 at 37 °C. Before the experiments, the cells were authenticated by short tandem repeat profiling and tested for mycoplasma contamination. For H/R conditions, Cells were exposed to a microaerophilic system (Thermo Fisher Scientific 8000, Marietta, GA) with 5% CO2 and 1% O2 balanced with 94% N2 at 37 °C for 6 h followed by reoxygenation in 95% air and 5% CO2 for 2 h to establish H/R conditions, as described in previous reports with a few modifications [28 (link)]. For 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP, Selleck), nicotinamide (NAM, Sigma-Aldrich) or trichostatin A (TSA, Sigma-Aldrich) treatment, the cells were incubated with 50 μM 3-TYP, 10 mM NAM or 0.5 μM TSA for the indicated times and then subjected to H/R, as needed [23 (link)].
3 typ
Manufactured by Selleck Chemicals
Sourced in United States, China
The 3-TYP is a laboratory equipment designed for general use in research and industrial settings. It serves as a versatile instrument capable of performing various analytical and experimental tasks. The core function of the 3-TYP is to facilitate controlled and precise operations within a laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
N acetyl cysteine (nac), by Selleck Chemicals (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (2 mentions)
S8628, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Apc h7 mouse anti human cd45, by BD (1 mentions)
Maml1, by Cell Signaling Technology (1 mentions)
P pi3k p85, by Cell Signaling Technology (1 mentions)
Senp1, by Cell Signaling Technology (1 mentions)
Acetylated sod2, by Abcam (1 mentions)
Sumo1, by Cell Signaling Technology (1 mentions)
Alexa fluor 647 rabbit anti active caspase 3, by BD (1 mentions)
T akt, by Cell Signaling Technology (1 mentions)
Ara c, by Merck Group (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Anti myc antibody sc 40, by Santa Cruz Biotechnology (1 mentions)
Anti hif 1α, by Cell Signaling Technology (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
10 protocols using 3 typ
1
In vitro model of alveolar epithelial cells
2
Investigating Apoptosis and Signaling Pathways
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Ara-C and DNR were purchased from the Sigma-Aldrich (St. Louis, MI, USA). NAC, Momordin-Ic and 3-TYP were consumed from Selleckchem (Houston, TX, USA). All compounds, except for in vivo studies, were reconstituted in the DMSO, stored at 100 mM stock concentrations in −80 °C, and used at the indicated doses as suggested by the vendor. Flow cytometry antibodies, Alexa Fluor 647 Rabbit anti-Active caspase 3 and APC-H7 Mouse anti-Human CD45 were purchased from BD pharmingen (San Jose, CA, USA). PE/Cy5 anti-Mouse CD45 (clone 30-F11) was consumed from BioLegend (San Diego, CA, USA). Immunoblotting antibodies, SUMO1, SIRT3, SENP1, Notch Activated Targets Antibody Sampler Kit including Notch1 (FL), MAML1, BPUSH and HES1, p-PI3K p85, t-PI3K p85, p-p38, t-p38, p-AKT and t-AKT were purchased from Cell Signaling Technology (Danvers, MA, USA). Acetylated SOD2 and SOD2 antibodies were purchased from Abcam (London, UK). Tubulin and β-actin antibodies were purchased from Proteintech (Rosemont, IL, USA).
3
Evaluation of Apoptosis and Oxidative Stress
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Anti‐Myc (#sc‐40) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti‐HIF‐1α (#36169) was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐β‐actin (#AC026) was purchased from ABclonal Company (Wuhan, China). Dual‐luciferase reporter assay system (#E194A) was purchased from Promega (Madison, WI, USA). 3‐TYP (#S8628) and NAC (#S1623) were purchased from Selleck (Houston, TX, USA). FITC‐Annexin V Apoptosis Detection Kit I (#556547) was purchased from BD Pharmingen (San Diego, CA, USA). CM‐H2DCFDA (#C6827) and MitoSOX™ Red (# M36008) were purchased from Thermo Fisher (Waltham, MA, USA). Annexin V‐FITC Apoptosis Detection Kit (#C1062) was purchased from Beyotime (Shanghai, China).
4
Evaluation of Small Molecule Compounds
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SU5416(CAS 204005–46-9) and Cell Counting Kit-8 (CCK-8) (CAS C0005) purchased from TargetMol, Trimetazidine (TMZ) was obtained from Servier (Tianjin). 3-TYP (S8628) was purchased from selleck.cn. β-Sitosterol (CAS 83–46–5), luteolin (CAS 491–70–3), Kaempferol (CAS 520–18–3), Costunolide (CAS 553–21–9), Naringenin (CAS 480–41–1),and Quercetin (CAS 117–39–5)were purchased from Chengdu Ruifensi Biotechnology Co., Ltd. (Sichuan, P. R. China).
5
Murine Sepsis Model and Interventions
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Male C57BL/6 mice (weighing 19–23 g, 6–8 weeks old) were obtained from the Animal Experimental Center of Southern Medical University. The experiments were approved by the Animal Care Committee of the Southern Medical University of China and were performed according to the National Institutes of Health guidelines for ethical animal treatment. The CLP procedure was performed as described in a previous protocol [57 (link)]. 5 mg/kg 3-TYP (S8628, Selleck), 25 mg/kg DCA (S8615, Selleck), 1 g/kg NaLa (71718, Sigma), and 20 mg/kg GSK (HY-100681, MCE) were intraperitoneally injected 0.5 h before CLP as needed. Mdivi-1 (50 mg/kg, S7162, Selleck) was intraperitoneally injected 1 h before CLP.
6
Apoptosis Regulation via PPAR-α Pathway
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Glucose was from Sigma-Aldrich (Shanghai, China). Annexin V-FITC apoptosis detection kit, CCK8 assay kit and cellular reactive oxygen species detection kit were obtained from Beyotime Biotechnology. 3-TYP and Wy14643 were from Selleck Chemicals, and GW6471 from R&D Systems China. TRIzol reagent and reverse transcription kits were obtained from Thermo Fisher Scientific, Inc. PPAR-α antibody was from Abcam. SIRT3, cleaved caspase-3, Bax, Bcl2, JNK1/2, phosphorylated JNK1/2 (p-JNK1/2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were from Cell Signaling Technology.
The study was approved by the Ethics Committee of Central Hospital of Minhang District (Shanghai, China).
The study was approved by the Ethics Committee of Central Hospital of Minhang District (Shanghai, China).
7
Sepsis Induction and Treatment in Mice
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C57BL/6 male mice (7–8 weeks) weighing 18–22 g were obtained from the animal lab center of Southern Medical University (Guangzhou, China). The animal experiments were complied with the ARRIVE guidelines and conducted in strict accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). The research programme was approved by the Ethics Committee of Animal Experiments of Southern Medical University. The sepsis mice was established using CLP method as described before [48 (link)]. All mice were anesthetized with pentobarbital (50 mg/kg) before the procedure. The chemical reagents with doses of 30 mg/kg melatonin (MCE, HY-B0075), 5 mg/kg 3-TYP (Selleck, S8628) and 50 mg/kg chloroquine (Selleck, S6999) were administered by intraperitoneal injection for consecutive 3 days before CLP. Mice were sacrificed 24 h after CLP for samples of blood and kidney tissue.
For survival study, mice were returned to their cages after awakening from anesthesia. 10 mice were included in each group and recorded every 2 h over a 5-day period. Apnea for more than 1 min was considered to indicate death. Mice that survived for more than 5 days were sacrificed by cervical dislocation.
For survival study, mice were returned to their cages after awakening from anesthesia. 10 mice were included in each group and recorded every 2 h over a 5-day period. Apnea for more than 1 min was considered to indicate death. Mice that survived for more than 5 days were sacrificed by cervical dislocation.
8
Molecular Inhibition of SIRT3 Pathway
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3-TYP, a selective inhibitor of SIRT3, was obtained from Selleck Chemicals (Shanghai, China). Actinomycin D was purchased from APE-Bio (Houston, USA). TSA and NAM were from MedChemExpress LLC (Shanghai, China). The expressing plasmids of vIL-6-Flag and vFLIP-Flag were generated as previously described [24 (link),59 (link)]. The skeleton plasmid for lentiviral vectors vCyclin-Myc, SERBP1-HA/Myc, SIRTs-Myc and Lipt2-Flag was pCDH-CMV-MCS-EF1-copGFP (referred to as pCDH). The expressing plasmid of LANA-Flag was kindly provided by Dr. Ke Lan from Wuhan University. The small guide RNA sequences (sgRNAs) targeting SERBP1 and SIRT3 are listed in S1 Table . The control vector of all sgRNAs was Lenti-CRISPR-V2 (Lenti-V2 for short). All constructs were verified through DNA sequencing.
9
Sirt3 Modulation in Adipocyte Differentiation
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3T3-L1 cells were treated with the Sirt3 inhibitor 3-TYP (3-(1H-1,2,3-triazol-4-yl) pyridine) (Selleckchem, Houston, TX, USA) at 50 µM and 100 µM, or with the Sirt3 activator Honokiol (Tocris, Minneapolis, MN 55413, USA) at 1, 5, and 10 µM or were untreated as control and differentiated into adipocytes by the adipocyte differentiation procedure described below. Honokiol and 3-TYP treatments were applied during the process of differentiation of adipocytes (day 1 to day 6).
10
Metformin and 3-TYP in Cell Culture
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Metformin was purchased from Sigma (St.Louis, USA). It was dissolved in dimethyl sulfoxide (DMSO) for the in vitro assay and was stored at a concentration of 1 M, diluted with Dulbecco's modified Eagle's medium (DMEM), and a solvent control with DMSO was performed at no more than 2‰ (v/v). 3-TYP was purchased from Selleck (Shanghai, China) and was dissolved in DMSO and stored at a concentration of 5 mM.
DMEM/F12 and fetal Bovine Serum (FBS) were from Gibco BRL (Grand Island, NY, USA). Antibodies against Parkin, LC3B and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology (Boston, USA). Antibodies against PINK1, MMP3, MMP13 and Collagen Ⅱwere from Abcam (Cambridge, UK). IL-1β was from Preprotech (Chicago,USA). Phenylmethylsulfonyl fluoride (PMSF), ethylenediamine tetraacetic acid (EDTA), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide (MTT) and other chemicals were purchased from Sigma.
DMEM/F12 and fetal Bovine Serum (FBS) were from Gibco BRL (Grand Island, NY, USA). Antibodies against Parkin, LC3B and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology (Boston, USA). Antibodies against PINK1, MMP3, MMP13 and Collagen Ⅱwere from Abcam (Cambridge, UK). IL-1β was from Preprotech (Chicago,USA). Phenylmethylsulfonyl fluoride (PMSF), ethylenediamine tetraacetic acid (EDTA), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide (MTT) and other chemicals were purchased from Sigma.
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