To determine the initial response triggered by osteoclastogenic stimulation, bone marrow-derived cells were pre-treated with 1 µmol·L−1 SB202190 (Tocris), 5 µmol·L−1 of FR180204 (Tocris), 1 µmol·L−1 SU6656 (Selleck Chemicals), 40 µmol·L−1 T-5224 (ApexBio Technology), or vehicle 2 hours before osteoclastogenic stimulation. Cells were lysed after 6 hours of RANKL + TNFα costimulation, and the Ogt and Oga mRNA levels were measured by quantitative real-time PCR.
T 5224
Manufactured by Apexbio
Sourced in United States
The T-5224 is a laboratory instrument designed for the analysis and quantification of biological samples. It utilizes advanced spectrophotometric technology to measure the concentration and purity of various biomolecules, such as proteins, nucleic acids, and small molecules, in solution. The T-5224 provides accurate and reliable data to support scientific research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Sp600125, by InvivoGen (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Sb202190, by Bio-Techne (1 mentions)
Fr180204, by Bio-Techne (1 mentions)
Su6656, by Selleck Chemicals (1 mentions)
Hec 1b, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Hec50b, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Il 23, by BioLegend (1 mentions)
Cl264, by InvivoGen (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Cl307, by InvivoGen (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Resiquimod, by InvivoGen (1 mentions)
Ll 37, by InvivoGen (1 mentions)
Rna40, by InvivoGen (1 mentions)
11 protocols using t 5224
1
Osteoclastogenic Stimulation Response
2
Characterizing Differentiation of Human EC Cell Lines
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Ishikawa, HEC-1B and HEC-50B human EC cell lines were all purchased from JCRB cell bank (Tokyo, Japan) and cultured according to manufacturer's instructions. Ishikawa is highly differentiated, HEC-1B is moderately differentiated and HEC-50B is poorly differentiated. For knockdown and overexpression experiments, cells were transfected with Lipofectamine 3000 (Thermo Fisher Scientific). We used cells transfected with blank vector plasmid as negative control group. T-5224 (AP-1 inhibitor, Catalog No. B4664) was purchased from APExBIO (Houston, USA).
3
Evaluating T-5224 Efficacy in Cell Cultures
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The cells were plated and incubated in DMEM or RPMI-1640 supplemented with 10% FBS, and 1% P/S for overnight. The culture media were replaced by DMEM or RPMI-1640 supplemented with 0.5% FBS, and 1% P/S. After 24 h, T-5224 (APExBIO Technology) was supplied in the culture media supplemented with 0.5% FBS, and 1% P/S for 48 h exposure in vitro experiments.
4
Generation of BM-derived Immune Cells from Mx1-Cre Mice
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To generate BM‐derived immune cells from Mx1‐Cre mice in vitro, deletion was first induced by poly I:C injection in vivo (as described above). Controls were treated equally. BM was then isolated from tibia and femur, and red blood cells were lysed. Isolated cells were cultured in 4 ml RPMI‐1640 (Gibco) with 10% FCS (PAA), penicillin, streptomycin, non‐essential amino acids (Sigma‐Aldrich), sodium pyruvate (Sigma‐Aldrich), β‐mercaptoethanol (Gibco), and GM‐CSF (20ng/mL, Peprotech) for 3 days. On day 3, media was changed. On day 6, non‐adherent cells (BMDCs) were harvested and used for experiments (CD11c+ cells > 80%). BMDCs were stimulated with IMQ, CL264, or CL307 (InvivoGen). For inhibition BMDCs were pretreated with SP600125 (InvivoGen; 25 µM) or T‐5224 (20 µM, ApexBio) for 1 h before stimulation. Protein levels of CCL2 (BD Biosciences) and IL‐23 (BioLegend) were analyzed by ELISA in BMDC supernatants. BM‐pDCs were generated as previously described (Drobits et al,2012 ).
5
Modulation of Dendritic Cell Responses
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Human mo‐DCs were pretreated for 1 h with JNK inhibitor SP600125 (25 µM, InvivoGen) or the AP‐1 inhibitor T‐5224 (20 µM, ApexBio) before a stimulation with 10 µg/ml of Resiquimod (R848, InvivoGen) or RNA‐40 (10 µg/ml; iba‐lifesciences) in complex with LL‐37 (50 µg/ml; InvivoGen) for 4 or 24 h. For complex formation, RNA‐40 was incubated with LL‐37 for 1 h at room temperature. Human CCL2 and IL‐23 (Thermo Fisher Scientific) was analyzed by ELISA.
6
Transwell Assay for HCC-HUVEC Interactions
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A transwell system was used for examining the interaction between HCC cell lines and HUVECs. The lower compartment of each 24-well transwell plate was coated with Matrigel. Each well was seeded with 4×104 HUVECs in Medium 200PRF (Thermo Fisher, Waltham, MA, USA). The upper compartment of each transwell plate was seeded with 1×104 HuH7-S2, HuH7-core-high, or HuH7-core-low cells in serum-free DMEM. The upper compartment was then placed onto the lower compartment and the transwell plates were incubated at 37°C in a 5% CO2 environment for 6 hours. Calcein AM fluorescent dye was added and incubation was continued for another 30 minutes. Next, the cells in the lower compartment were examined under a microscope and quantitatively analyzed using MetaMorph (Molecular Devices, Sunnyvale, CA, USA). For validating the effects of VEGF, 1 mg/mL bevacizumab (Roche, Switzerland) or control immunoglobulin G1 (IgG1) was added to the culture media of both the HCC cells and HUVECs. For validating the effects of AP-1 inhibition, 50 μM of an AP-1 inhibitor, T-5224 (Apexbio, Houston, TX, USA), was added to the culture media of both the HCC cells and HUVECs.
7
Drug Treatment and Cell Line Analysis
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The human leukemia (HEL 92.1.7, K562) and epithelial-like HEK293T (CRL-3216) cell lines were obtained from ATCC (US) and tested negative for mycoplasma. These cell lines were cultured and maintained in Dulbecco’s Modified Eagle Medium supplemented with HyClone 5% fetal bovine serum (GE Healthcare, US).
For drug treatment, cells were treated with Camptothecin (MedChemExpress, CN), T5224 (APExBIO, CN) and R406 (Beyotime Biotechnology, CN) for indicated times and used for cell proliferation analysis. Generation of K562-fli1 cells was previously described [21 (link)]. For FLI1 induction, cells were treated with 5μM of doxycycline (Solarbio, CN).
For drug treatment, cells were treated with Camptothecin (MedChemExpress, CN), T5224 (APExBIO, CN) and R406 (Beyotime Biotechnology, CN) for indicated times and used for cell proliferation analysis. Generation of K562-fli1 cells was previously described [21 (link)]. For FLI1 induction, cells were treated with 5μM of doxycycline (Solarbio, CN).
8
Cl-amidine and Small Molecule Inhibitors
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Cl-amidine (Calbiochem, Darmstadt, Germany), the TLR4 agonist lipopolysaccharide (ultrapure LPS; Invivogen, San Diego, CA, USA), SB203580, U0126, SP600125, BAY 11-7085, PI-103 (Adooq, Irvine, CA, USA), Tofacitinib, niclosamide (Tocris, Bristol, UK), and T-5224 (APExBIO, Houston, TX, USA) were purchased commercially. Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ten or twenty millimoles stock solution of Cl-amidine was dissolved in phosphate buffer solution (PBS), and other inhibitors were dissolved in DMSO. PBS or DMSO were used as vehicle controls in each experiment.
9
Bacillus and Staphylococcus Strain Acquisition
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B. subtilis ATCC 6633 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). B. subtilis KCTC 6633, KCTC 3014, KCTC 3135, KCTC 3239, and Bacillus cereus KCTC 13153 were purchased from the Korean Collection for Type culture (KCTC; Daejeon, Korea). Staphylococcus aureus USA300 was obtained from the Nebraska Transposon Mutant Library (Omaha, NE, USA). DNase I was purchased from Roche Molecular Biochemicals (Laval, QC, Canada). Thiazolyl blue tetrazolium bromide (MTT reagent), JNK V inhibitor, proteinase K, and lipoprotein (LPP) lipase from Pseudomonas sp. were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antihuman TLR2 antibody and its isotype control antibody were purchased from Invivogen (San Diego, CA, USA). The inhibitors of ERK (PD98059), JNK (JNK inhibitor V), and p38 MAP kinase (SB203580) were purchased from Calbiochem (San Diego, CA, USA). APC anti-human TLR2 antibody and its isotype control antibody were purchased from Biolegend (San Diego, CA, USA). T-5224, an AP-1 inhibitor, was obtained from ApexBio Technology (Boston, MA, USA), and BAY11-7082, a NF-κB inhibitor, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Tryptic soy broth (TSB) was purchased from BD Biosciences (San Diego, CA, USA). All other materials were from Sigma-Aldrich unless stated otherwise.
10
Intrathecal Injection of Microglial and Astroglial Inhibitors
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Reagents and antibodies were obtained from the following suppliers: c-Fos/AP-1 inhibitor T-5224, PKCε inhibitor peptide, and STAT3 inhibitor APTSTAT3-9R (Apexbio Technology LLC., TX, USA); specific microglia inhibitor minocycline hydrochloride (Mino) and astroglial toxin L-2-aminoadipic acid (LAA; Sigma–Aldrich Corp., MO, USA); and rat IL-6 and anti-IL-6 antibodies (PeproTech Inc., NJ, USA and Abcam, Cambridge, UK, respectively). Anti-IL-6 and Mino were diluted with phosphate-buffered saline (PBS) and saline, respectively; the other agents were dissolved in 1% dimethyl sulfoxide (DMSO). The aforementioned chemicals (50 μL) were injected intrathecally via the L5-6 lumbar interspace identified by the tail flick reflex [17 (link)] under 1%–3% isoflurane (Baxter, IL, USA) anesthesia delivered at an oxygen flow rate of 1 L/min.
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