Tak 242

Raw264.7 macrophages were obtained from the Shanghai cell bank of the Chinese Academy of Sciences. Raw264.7 macrophages were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies, Waltham, MA, USA). The cells, induced to differentiate as previously described, were stimulated with 100 U/ml murine recombinant IFNγ (PeproTech, Rocky Hill, NJ, USA) or 20 ng/ml murine recombinant IL-4 (PeproTech, Rocky Hill, NJ, USA) for 24 h to induce M1 or M2 polarization, respectively [23 (link)].
After Raw264.7 cells were induced into M1 or M2 macrophages, the cells were pre-treated with TAK-242 (1 μM; MedChemExpress, Monmouth Junction, NJ, USA) or deionized water (PeproTech, Rocky Hill, NJ, USA) as vehicle control for 1 h and were continuously treated with relaxin (100 ng/ml; 16.8 nmol/l) for 72 h. Cells treated with deionized water or TAK-242 alone (1 μM) for 72 h were used as appropriate controls.

The LMWF was obtained from New Zealand Undaria pinnatifida as described previously [17 (link)]. RPMI-1640 medium and phosphate-buffered solution (PBS) were bought from Gibco (Gaithersburg, MD, USA). Lipopolysaccharides (LPS), polymyxin B (PMB), and FITC-DEXTRAN (42,000 Da) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Granulocyte-macrophage colony-stimulating factor (GM-CSF) and TAK-242 were purchased from PeproTech (Rocky Hill, NJ, USA) and Medchemexpress (Monmouth Junction, NJ, USA), respectively.

Raw264.7 and A549 cells were purchased from Korea Cell Line Bank (Korea) and Transfected-(OPRP⁺), untransfected-Raw264.7 (OPRP⁻), and A549 cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Waltham, MA, USA) at 37 °C in a culture flask with an atmosphere containing 5% CO₂. TLR4 inhibitor, TAK-242 (Invitrogen), IFN-α (Peptron, Daejeon, Korea), IFN-β (Peptron), and IFNAR2 (Abcam, Cambridge, UK) were purchased for scrutinizing cell signaling.

HO-8910PM cells were seeded in 96-well plates (2.0×10⁴ cells/well). After 12 h of incubation, experimental group was stimulated with 0.1 μg/ml LPS (Sigma, Santa Clara, CA, USA) dissolved in Phosphate Buffered Saline (PBS), while LPS was replaced by serum-containing medium in control group. The proliferation assay was done with 3 - (4, 5 – dimethylthiazol – 2 - ylx) - 2, 5 - diphenyltetrazolium bromide (MTT). 200 μl of 0.5 mg/ml MTT was added to the medium, and the cells were incubated at 37°C for 4 h. Then the culture medium was discarded and 150 μl DMSO (Invitrogen) was added to each well to dissolve the precipitate. The OD at 492 nm was measured at indicated time periods (0 h, 2 h, 4 h, 6 h, 8 h, 12 h) after stimulation using the Microplate Reader (Rayto, Shenzhen, China).
In seperate experiments, cells were divided into four groups: the control group (C) and LPS group (L) were pretreated with serum-containing medium, while the inhibitor group (I) and inhibitor+LPS group (I+L) were pretreated with 1 μg/ml TLR4 inhibitor TAK-242 (Invitrogen) instead. After 6 h of pretreatment at 37°C, culture medium was discarded in all groups. Then serum-free medium was added to C and I group, LPS (Sigma) was added to L and I+L group. All the groups were maintained in culture for another 6 h. MTT assay was performed as described above.

Alginate, LPS, PMB, FITC-phalloidin, FITC-LPS and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), penicillin and streptomycin were purchased from Hyclone (Logan, UT, USA). Foetal bovine serum (FBS) was obtained from Biological Industries (Beit-Haemek, Israel). Anti-mouse TLR4, MD2, MyD88, F4/80 and Ly6G antibodies were from purchased Abcam (Cambridge, UK). Antibodies against Akt, phosphor-Akt (p-Akt), NF-κB p65, IκB, phosphor-IκB (p-IκB), mTOR, phosphor-mTOR (p-mTOR), p70 S6 kinase (p70 S6K), phosphor-p70 S6K (p-p70 S6K), p38, phosphor-p38 (p-p38), JNK, phosphor-JNK (p-JNK), ERK, phosphor-ERK (p-ERK) and iNOS were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin, -β-tubulin, -GAPDH and -Lamin B1 antibodies were purchased from Proteintech (Hubei, China). Inhibitors LY 294002, SB 20358, SP 600125 and PD 98059 were obtained from Selleck (Shanghai, China) and TAK-242 and rapamycin from Invitrogen (Carlsbad, CA, USA). The other chemicals were all purchased from Macklin Biochemical Technology (Shanghai, China).