The chemical formula of derivative 4F is C35H33N2O6 (1, 8-bis (benzyloxy)-3-(4-(2-hydroxyethyl) piperazine-1-carbonyl)-9,10-anthraquinone) ( Figure 1B ) , with a relative molecular mass of 577 and >98% purity [high-performance liquid chromatography (HPLC)]. Derivative 4F is a yellow flaky crystal that is slightly soluble in water and soluble in organic solvents such as dimethyl sulfoxide (DMSO), with a melting point of 155–157°C. Rhein was purchased from Langze (Nanjing, China), vincristine (VCR) was purchased from Wanle (Shenzhen, China), paclitaxel (PTX) was purchased from Yangzijiang (Jiangsu, China), cisplatin (DDP) was purchased from Haosen (Jiangsu, China), and Rac1 activator PMA and Rac1 inhibitor NSC23766 were purchased from Selleck (Houston, USA).
Phorbol myristate acetate (pma)
Manufactured by Selleck Chemicals
Sourced in United States
The PMA is a high-quality laboratory instrument designed for chemical analysis and research applications. It is a precise and reliable device used for the measurement and monitoring of various chemical parameters. The core function of the PMA is to provide accurate and consistent data to support scientific investigations and experiments.
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Lab products found in correlation
Nsc23766, by Selleck Chemicals (1 mentions)
Interleukin 4 (il 4), by Proteintech (1 mentions)
Ifn γ, by Proteintech (1 mentions)
Sq22536, by MedChemExpress (1 mentions)
Forskolin, by MedChemExpress (1 mentions)
Sodium oxamate, by Selleck Chemicals (1 mentions)
U73122, by Merck Group (1 mentions)
Aktviii, by Merck Group (1 mentions)
Lapatinib, by Selleck Chemicals (1 mentions)
Tak 632, by Selleck Chemicals (1 mentions)
Mouse anti hsc70 sc 7298, by Santa Cruz Biotechnology (1 mentions)
Gf109203x, by Selleck Chemicals (1 mentions)
Fr180204, by Selleck Chemicals (1 mentions)
Ruxolitinib, by Selleck Chemicals (1 mentions)
Zstk474, by Selleck Chemicals (1 mentions)
Cycloheximide (chx), by Selleck Chemicals (1 mentions)
U0126, by Selleck Chemicals (1 mentions)
Thp 1, by Solarbio (1 mentions)
Raw264, by Solarbio (1 mentions)
Human ifn γ, by Thermo Fisher Scientific (1 mentions)
9 protocols using phorbol myristate acetate (pma)
1
Synthesis and Characterization of Anthraquinone Derivative 4F
2
Macrophage Differentiation and Polarization
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THP-1 cells were differentiated into intermediate-stage M0 cells by treatment with 300 nM phorbol 12-myristate 13-acetate (PMA, #S7791, Selleckchem) for 48 h. Subsequently, M1 and M2 macrophages were induced with 20 ng/mL IFN-γ (#AF-345-05-20 µG, Proteintech) for 48 h and 20 ng/mL IL-4 (#AF-214-14-5 µG, Proteintech), respectively, for 48 h on the basis of M0 macrophages. For ESCC TAMs, M0, M1, and M2 macrophages were coincubated with conditioned medium produced by ESCC cells. Note: THP-1 cells are oval suspension cells, M0 TAMs grow adherently but are oval in shape, M1 TAMs are irregular in shape with multiple radial antennae, and M2 TAMs are fusiform with elongated antennae on both sides. These morphological features were used to observe macrophages after treatment with conditioned media.
3
Modulating Metabolic Pathways in HMGB1 Signaling
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Compounds and reagents include: lactate (#L6402, Sigma-Aldrich, USA), phorbol 12-myristate 13-acetate (PMA, #S7791, Selleck Chemicals, USA), LDHA inhibitor, sodium oxamate (SO, #6871, Selleck Chemicals, USA), MCT inhibitor, a-cyano-4-hydroxycinnamate (CHC, #S8612, Selleck Chemicals, USA), HMGB1 inhibitor, glycyrrhizin (#HY-N0184, MedChemExpress, USA), adenylate cyclase inhibitor, SQ22536 (#HY-100396, MedChemExpress, USA), PKA inhibitor, H89 (#HY-15979, MedChemExpress, USA), Forskolin (#HY-15371, MedChemExpress, USA), rHMGB1 (#ab167718, Abcam, USA).
4
Signaling Pathway Protein Detection
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Rabbit anti-PHLDA1 (ab133654) was from Abcam (Cambridge, UK). Mouse anti-HSC70 (sc-7298) was from Santa Cruz (Heidelberg, Germany). Mouse anti-AKT (9220S), mouse anti-ERK1/2 (4696S), rabbit anti-p-AKT (Ser473) (9271S), rabbit anti-p-ERK (Thr202/Tyr204) (9101S), rabbit anti-acetyl-histone H3 (Lys27) (8173P), rabbit anti-histone H3 (9715), rabbit p-STAT3 (Tyr705) (9145S), and mouse anti-STAT3 (9139) were from Cell Signalling Technology (Leiden, Netherlands). Lapatinib, ZSTK474, GF109203X, TAK632, ruxolitinib, FR180204, PMA, and 4SC-202 were purchased from Selleckchem (Houston, TX, USA). U73122 and AKT-VIII were purchased from Sigma.
5
Activation of Human Mast Cell Line
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The human MC line HMC-1 was obtained from the ATCC and were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% P/S. For HMC-1 activation, the cells (1 × 106/ml) were stimulated with phorbol 12-myristate 13-acetate (PMA, 20 nM, Selleck, USA) and calcium ionophore (A23187, 1 μM, Aladdin, China) (PMACI) (34 (link)) for 16 h. The cell supernatants were collected for ELISA analysis, and the cells were collected for real-time PCR and Western blot analysis.
6
U937 Macrophage-Derived Foam Cell Model
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Macrophages differentiation from U937 cells in vitro were induced by treatment with 100 ng/ml PMA (Selleck, S7791) for 24 h, and macrophage‐derived foam cell model was generated by incubation with 100 μg/mL ox‐LDL for 24 h.
7
Inhibition of Cellular Signaling Pathways
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Sorafenib (MCE, USA) was dissolved in DMSO, the stock solutions (90 mM) were stored at –80 °C in single-dose vials, avoiding repeated freeze-thaw. Dilute the stock solutions with PBS to a working Solution (90 nM) for inhibiting VEGFR2 according to the user instruction. GW4869 (MCE, USA) was stored at −80 °C as a 1.5 mM stock suspension in DMSO, avoiding repeated freeze-thaw. Dilute the stock solutions with PBS to a concentration of 20 μM for inhibition of exosome release. CHX (Selleck, USA) was dissolved in DMSO and stored at –80 °C in single-dose vials and diluted to a working solution (20 μg/ml) for inhibiting protein synthesis. MG132 (MCE, USA) was dissolved in DMSO and stored at –80 °C in the stock solutions(10 mM), diluted to a working Solution (10 μM) for inhibiting the proteolytic activity of the 26S proteasome complex. U0126 (Selleck, USA) was dissolved in DMSO and diluted to a working Solution (10 μM) for inhibiting MEK1 and MEK2. PMA (Selleck, USA) was dissolved in DMSO and diluted to a working solution (100 nM) for activating ERK phosphorylation.
8
Validated Cell Lines for Colon Cancer Research
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We obtained murine colon carcinoma cell line CT26 from the National Collection of Authenticated Cell Cultures (NCACC). Cells were cultured in RPMI1640 medium (Solarbio, 31800) supplemented with 10% FBS (Solarbio, S9030). Murine MC38 colon cancer cell line was from BNCC and cultured in DMEM (Solarbio, 12100) with 10% FBS (Solarbio, S9030). Human monocytic cell line THP-1 was from NCACC and cultured with RPMI1640 medium (Solarbio, 31800) supplemented with 10% FBS (Solarbio, S9030). When indicated, THP-1 monocytes were incubated with phorbol 12-myristate 13-acetate (PMA; 100 ng/mL; Selleck, S7791) for 24 hours to differentiate into macrophages. Murine macrophage cell line RAW264.7 was from NCACC and cultured in DMEM (Solarbio, 12100) with 10% FBS (Solarbio, S9030). All cell lines were cultured at 37°C in a humidified atmosphere with 5% CO2. Authentication for all cell lines was acquired from the manufacturers and reauthenticated with short tandem repeat test within the past year. Cells were cultured for approximately 3 months for experiments and were regularly tested for Mycoplasma with PCR method. No more than 10 passages for THP-1 and RAW264.7 cells and no more than 20 passages for CT26 and MC38 cells were used.
9
Murine Anti-Inflammatory Cytokine Assay
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All experiments were performed with the approval of the Ethics Committee and in accordance with the guidelines of the Institutional Animal Care and Use Committee of Gyeongsang National University (GNU-160811-M0036). Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). Stock solutions of colivelin (378 µM; Tocris Bioscience, Bristol, UK), doxorubicin (2 mg/mL), and lipopolysaccharide (LPS; 5 mg/mL) were prepared in distilled water. The mouse IL-4 (0.1 mg/mL), human IL-4 (0.1 mg/mL), human IL-13 (0.1 mg/mL), and human IFN-γ (0.1 mg/mL), which were purchased from PeproTech (Rocky Hill, NJ, USA), were dissolved in distilled water. Bay 11-7085 (50 mM), necrostatin-1 (20 mg/mL; Cayman Chemical, Ann Arbor, MI, USA), PMA (1 mM), SB203580 (25 mM), S3l-201 (50 mM; Selleckchem, Houston, TX, USA), Z-VAD-FMK (10 mg/mL; InvivoGen, San Diego, CA, USA), and Z-YVAD-FMK (20 mM; Abcam, Cambridge, MA, USA) were dissolved in dimethyl sulfoxide (DMSO). U-46619 (28.53 mM) was prepared in methyl acetate. The solutions were then diluted in culture medium to the working concentration. An equivalent concentration or volume of solvents was added in the control group. The final concentrations of DMSO and methyl acetate were ~0.1% (v/v).
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