HaCaT cells were obtained from Cell Bank of the Chinese Academy of Sciences in Beijing, China. Primary mouse keratinocyte (MKs) were cultured using a described method before 23 (link). Briefly, neonatal Balb/c mice (postnatal day 1-3) mice were immersed in 75% alcohol for 2-3 minutes, and then rinsed with PBS for 2-3 times. Gradually blunt skin separation with scissors and tweezers. After repeated washed by PBS, adding neutral protease II (1x) until submerged the skin, 4°C for the night. After the epidermal and dermis were separated, the epidermis was cut in sterile PBS, digested with 0.25% trypsin/ 0.02% EDTA solution for 2-3 minutes, then neutrallized with 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin, and filtered with a sieve after blowing, centrifugated for 5min. MKs can be inoculated after resuspending with 1640 medium. Both HaCaT and primary mouse keratinocytes were cultured in 1640 medium (SH30809.01B, Hyclone, USA) containing 10% fetal bovine serum (S-FBS-500, Scitecher, USA) and 1% penicillin/ streptomycin (GA3502, Genview, Australia), digested with 0.25% trypsin/0.02% EDTA solution (SH30042.01, Hyclone, America). Cell cultures were performed in a 5% CO2 atmosphere at 37°C. Cells were treated with 1mM AICAR (ab120358, Abcam, UK), 2μM Compound C (ab120843, Abcam, UK).
Ab120843
Manufactured by Abcam
Sourced in United Kingdom
Ab120843 is a lab equipment product. It is a recombinant antibody specific for a target protein. This antibody can be used for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab120358, by Abcam (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ga3502, by Genview (1 mentions)
Endothelial cell growth supplement (ecgs), by Merck Group (1 mentions)
D150959, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem f12, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Ab3759, by Abcam (1 mentions)
Ab56788, by Abcam (1 mentions)
Ab104224, by Abcam (1 mentions)
Ab131357, by Abcam (1 mentions)
Ecl western blotting kit, by Solarbio (1 mentions)
H 600 transmission electron microscope, by Hitachi (1 mentions)
Total protein extraction kit, by Keygen Biotech (1 mentions)
Fx 5000 system, by Flexcell (1 mentions)
12 protocols using ab120843
1
Isolation and Culture of Keratinocytes
2
Metformin Attenuates Hypoxia-Reoxygenation Injury
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The NRVMs were randomly divided into four groups: i) Control (CON) group, cells were incubated with normal oxygen; ii) hypoxia-reoxygenation (HR) group, in which the cells were incubated in hypoxic conditions for 4 h and reoxygenated for 6 h; iii) HR + metformin (HR + met) group, at the onset of reoxygenation, metformin (0.1 mM) was added to the DMEM (the dose of metformin used in the present study was selected based on a previous study) (26 (link)); and iv) HR + metformin + compound-C (HR + met + compound-C), at the onset of reoxygenation, metformin and compound-C (10 nM; cat. no. ab120843; Abcam) were added into the medium simultaneously (the dose of compound-C used in the present study was selected based on a previous study) (26 (link)).
3
Ovarian Cancer and Endothelial Cell Cultures
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The ovarian cancer cell lines SKOV3 and UCI101 were maintained in Dulbecco's modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, California, USA). HUVEC were isolated from umbilical cords obtained with patient consents and approved by the ethical committee at the Hospital Clínico Universidad Católica de Chile. HUVEC were obtained by collagenase treatment and maintained in Human Endothelial SFM medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and endothelial cell growth supplement (6 mg/ml final concentration) (Merck Millipore, Billerica, MA, USA). These cells were used until passage five. Endothelial EA.hy926 cells were maintained in Iscove's Modified Dulbecco's Media (IMDM) supplemented with 10% fetal bovine serum. For all experiments, endothelial cells were used until passage six. Metformin was solubilized in water (D150959; Sigma-Aldrich, St. Louis, MO, USA), and Compound C (Dorsomorphin) was solubilized in DMSO (ab120843; Abcam, Cambridge, UK).
4
Mitochondrial Function Modulation Assay
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AMPK activator AICAR (2 mM, ab120358) and AMPK inhibitor compound C (20 μM, ab120843) were purchased from Abcam. Respiratory inhibitors rotenone (0.5 μM) or antimycin A (1 μM) and ATP synthase inhibitor oligomycin (1 μM) were purchased from Sigma-Aldrich. MitoTracker Red CMXRos (100 nM, M7512) and MitoTracker Deep Red FM (100 nM, M22426) were purchased from Molecular Probes. Final concentration of dimethyl sulfoxide was set to 0.5% during the treatment with compound C, rotenone, antimycin A, or oligomycin. The primary and secondary antibodies used for immunoblotting or immunostaining are listed in Supplementary Table 2 . Additional information of materials and antibodies are shown in Supplementary Methods .
5
Mitochondrial Dynamics and AMPK Signaling in Neuronal Injury
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The following materials were used in this study including enhanced chemiluminescence (ECL) Western blotting kit (Solarbio, Beijing, China), horseradish peroxidase-conjugated rabbit anti-goat IgG and goat anti-rat IgG (Pierce, USA), rabbit anti-rat CaMKK (ab80066, Abcam, UK), rabbit anti-rat AMPKα (ab3759, Abcam, UK), rabbit anti-rat pAMPKα (ab131357, Abcam, UK), rabbit anti-rat Drp1 (ab56788,Abcam,UK), rabbit anti-rat NIX and BNIP3 IgG (D4R4B, CST, USA), mouse anti-rat NeuN (ab104224, Abcam, UK), rabbit anti-rat STO609 (ab141591, Abcam, UK), rabbit anti-rat Dorsomorphin/Compound C (ab120843, Abcam, UK), a fluorescence microscope X81 (Olympus, Tokyo, Japan), total protein extraction kit (Keygen Biotech, Nanjing, China), and Transmission Electron Microscope H-600 (Hitachi, Japan).
6
Biaxial Strain on Chondrocytes
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Chondrocytes were grown on collagen I‐coated Bioflex 6‐well culture plates (Flexcell International, Hillsborough, NC) to 80%‐90% confluence. CTS experiments were performed using the FX‐5000 Flexcell system (Flexcell International, McKeesport, PA). To provide uniform radial and circumferential strain on the membranes, the plates were placed on a loading station (located in an incubator with 5% CO2) such that when a vacuum was applied to the loading station, the membrane deformed across the post face, creating uniform biaxial strain. Chondrocytes were subjected to CTS (10%, 0.5 Hz) for different durations (0, 0.5, 1, 2, 4, 8, and 16 hr) with or without IL‐1β for 24 hr. The stimulations of CTS and IL‐1β on chondrocytes began at the same time. We choose the best condition for further study. Compound C (ab120843; Abcam), a selective and reversible AMPK inhibitor, was used for pretreatment for 1 hr before the stimulation with IL‐1β and CTS (Dai et al., 2017 ).
7
Normal and Cancer Cell Culture
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Normal human fibroblasts isolated from healthy newborn foreskins and provided by Dr. Jin-Ho Chung (Seoul National University, Korea) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1 g/L glucose (12-707F, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) (S-FBS-US-015, Serana, Bunbury, WA, Australia) and glutamine (25030-081, Gibco, Waltham, MA, USA). Human cancer lines MCF-7 and HCT116 were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose (12-107F, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) (S-FBS-US-015, Serana, Bunbury, WA, Australia) at 5% CO2 and 37 °C. For glucose withdrawal, DMEM free of glucose (LM001-79, Welgene, Daegu, Korea) was used. Cells were treated with 5 mM N-acetylcysteine (NAC) (A7250, Sigma-Aldrich, St. Louis, MO, USA), 10 μM EX527 (E7034, Sigma-Aldrich), 2 μM compound C (ab120843, Abcam, Cambridge, UK), 5 mM 3-methyladenine (M9281, Sigma-Aldrich), and 250 nM Torin1 (orb146133, Biorbyt, Cambridge, UK). Other chemicals were purchased from Sigma-Aldrich unless stated otherwise.
8
Investigating Neurological Outcomes in ICH
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Animals that died or had a neurological deficit score higher than 20 at 24 h after surgery were euthanized and excluded from the analysis. A power analysis based on our previous studies65 (link) and pilot data indicated that eight mice/group would provide at least 80% power for detecting a 20% decrease in neurologic deficit at α = 0.05 (two-sided) in neurobehavior tests. The animals and cell cultures for each group were randomized. Treatment, data collection, and data analyses were blinded by using different investigators or by masking the sample labels.
For in vitro experiments, we added 20 μM hemin (7084–24–4, Frontier Scientific, USA) and/or 20 μM DCPIB (1540/10, Tocris, USA) to BV2, RAW 264.7, primary macrophages and microglia for 6 h. We treated cells with 5 μM ML385 (S8790, Selleck, USA), 10 μM Compound C (ab120843, Abcam, USA), anti-IgA (1:500, ab37322, Abcam), or anti-CD36 (1:500, ab23680, Abcam) 2 h before hemin and/or DCPIB exposure.
For in vivo experiments, 10 or 30 mg/kg DCPIB or vehicle was administered daily by intra-peritoneal injection to wildtype mice for 3 days beginning 2 h after ICH. Vehicle, 50 μM ML385, 100 μM compound C, anti-IgA (1:50), or anti-CD36 (1:50) was injected into the ipsilateral lateral ventricle of Lrrc8a conditional knockout (cKO) mice just before ICH.
For in vitro experiments, we added 20 μM hemin (7084–24–4, Frontier Scientific, USA) and/or 20 μM DCPIB (1540/10, Tocris, USA) to BV2, RAW 264.7, primary macrophages and microglia for 6 h. We treated cells with 5 μM ML385 (S8790, Selleck, USA), 10 μM Compound C (ab120843, Abcam, USA), anti-IgA (1:500, ab37322, Abcam), or anti-CD36 (1:500, ab23680, Abcam) 2 h before hemin and/or DCPIB exposure.
For in vivo experiments, 10 or 30 mg/kg DCPIB or vehicle was administered daily by intra-peritoneal injection to wildtype mice for 3 days beginning 2 h after ICH. Vehicle, 50 μM ML385, 100 μM compound C, anti-IgA (1:50), or anti-CD36 (1:50) was injected into the ipsilateral lateral ventricle of Lrrc8a conditional knockout (cKO) mice just before ICH.
9
Investigating the AMPK Signaling Pathway
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Human lung cancer cell lines H1299, A549, human breast cancer cell lines MCF-7, MDA-MB-231 were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM supplemented with 10% FBS and 1% antibiotic. Metformin (ab120847), AICAR (ab120358) and Compound C (ab120843) were purchased from Abcam (Shanghai, China). Antibodies against AMPKα1 (2795S), AMPKα2 (2757S), AMPKα (2532S), Phospho-AMPKα (Thr172) (2531S), PKCγ (43806S), Na, K-ATPase (3010S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against Phospho-PKCγ (Thr514) (AF8347) was purchased from Affinity (Cincinnati, OH, USA). Antibodies against Phospho-serine/threonine (ab15556) and β-actin (ab8227) were purchased from Abcam. Antibody against Hsp90α and recombinant Hsp90α were provided by Protgen (Beijing, China). Detailed information about the antibodies is shown in Table S3 (Supplementary Material) .
10
AMPK Modulation in Hepatic Carcinogenesis
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Male C57BL/6 mice were obtained from Chongqing Medical University. To induce hepatic carcinogenesis, 4-week-old C57BL/6 male mice received 75 mg/kg DEN (Sigma-Aldrich, N0258) in two divided doses and CCL4 (mixed with olive oil in a 1:4 ratio, 0.08 ml/10 g) twice a week until 20 weeks. After 8 weeks, fresh liver tissues were obtained through liver biopsy. Immunohistochemical staining was used to detect the level of AMPK phosphorylation. The same criteria with human tissue microarrays were adopted to distinguish the level of p-AMPK expression. In mice with p-AMPK high expression, dorsomorphin (Abcam, ab120843, 0.2 mg/kg d) was used to inhibit p-AMPK, or PBS as a control. In mice with p-AMPK low expression, metformin (Sigma, PHR1084, 250 mg/kg d) or AICAR (Sigma, A9978, 2.5 mg/kg d) were used to activate AMPK. For autophagy inhibition, mice received 50 mg/kg of chloroquine (Santa Cruz Biotechnology, sc-205629) via intraperitoneal injection once every 3 days along with a concurrent application of metformin. All mice were killed at 32 weeks. All animal experiments complied with the ARRIVE guidelines and was carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines.
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