Supplementary Materialsbiomolecules-10-00238-s001. using the TRIF-mediated complex formation composed of TRAF3, TANK, and IKK leading to downregulation of AKT phosphorylation and reduction of IRF-3 activation, resulted in inhibition of IRF-3-dependent expression of genes including 0111:B4) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Adenosine 5-triphosphate (ATP) was purchased from New England Biolabs, Inc. (Ipswich, MA, USA). Roswell Park Memorial Institute (RPMI) 1640, Dulbeccos altered Eagles medium (DMEM), penicillin-streptomycin, and trypsin were purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Biotechnics Research, Inc. (Irvine, CA, USA). TRIzol reagent was purchased from MRCgene (Cincinnati, OH, USA). Phosphate-buffered saline (PBS) was purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Phosphospecific or total-protein antibodies raised Fumaric acid against IRF-3, -actin, TBK1, IKK, AKT, AKT1, AKT2, lamin Fumaric acid A/C, Flag, and HA were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-TRAF3 antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). primers utilized for the semiquantitative reverse transcriptase (RT)-polymerase chain reaction were purchased from Bioneer Corp. (Daejeon, Korea). Additional primers used in this study were purchased from Macrogen Inc. (Seoul, Korea). PCRBIO HS Taq PreMix and qPCRBIO SyGreen Blue Mix Lo-ROX for PCR were purchased from PCR Biosystems Ltd. (London, UK). Constructs for signaling molecules such as Flag-TBK1-WT, HA-AKT1, and HA-AKT2 Fumaric acid were used as reported previously [18,19]. pcDNA3-IKK-Flag was a gift from Tom Maniatis (Addgene plasmid #26201, http://n2t.net/addgene:26201 RRID:Addgene_26201) [20]. Flag-TBK1-CC (TBK1 plasmid DNA mutant of the CC domain name) was constructed by a standard cloning method. All constructs were confirmed by automated DNA sequencing. RAW264.7 cells (ATCC number TIB-71) and HEK293T cells (ATCC number CRL-1573) were purchased from your American Type Culture Collection (ATCC) (Rockville, MD, USA). 2.2. Cell Culture and Compound Preparation A BALB/c-derived murine macrophage cell collection (RAW264.7) was cultured in RPMI 1640 media supplemented with 10% heat-inactivated FBS, 100 U/mL of penicillin, 100 g/mL of streptomycin, and 2 mM l-glutamine. A human embryonic kidney cell collection (HEK293T) was cultured in DMEM Fumaric acid media supplemented with 5% heat-inactivated FBS, 100 U/mL of penicillin, 100 g/mL of streptomycin, and 2 mM l-glutamine. Both cell lines were produced at 37 C under 5% CO2 in a humidified incubator. The stock answer of 8-HD was prepared by dissolving the 8-HD powder in 100% DMSO in a microcentrifuge tube. The use of DMSO treatment in the following study is in the same concentration as DMSO content in the diluted compound (8-HD). 2.3. Cell Viability Assay The cytotoxic effect of 8-HD on tested cells (RAW264.7 and HEK293T cells) was evaluated by conventional MTT assay as reported previously [21]. For instance, cells (105 cells/well) were plated in 96-well plates and incubated overnight, followed by 8-HD (0, 6.25, 12.5, 25, and Rabbit Polyclonal to FUK 50 M) treatment for 24 h. Next, 10 L of MTT answer (10 mg/mL in PBS pH 7.4) was added to the cell culture for 3 h at 37 C. The reaction then stopped by adding 100 L quit answer (15% sodium dodecyl sulfate), followed by incubation for 8 h at 37 C. The absorbance was then measured Fumaric acid at 570 nm using a Synergy HT Multi-Mode Microplate Reader (BioTek Devices GmbH, Bad Friedrichshall, Germany). 2.4. mRNA Expression Analysis by Semiquantitative Reverse Transcriptase (RT)-Polymerase Chain Reaction (PCR) and Quantitative Real-Time PCR (qPCR) RAW264.7 cells (106 cells/well) were pre-incubated overnight, followed by incubation with 8-HD (0, 12.5, 25, and 50 M) for 30 min and additional incubation with LPS (1 g/mL) for 6 h or.
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