Recombinant mouse tnf α

Brazilin used in this study was isolated from the dried heartwood of Caesalpinia sappan L as described previously [30 (link)]. All commercial antibodies and chemicals were purchased from the following resources: Anti-IκB-α, anti-phospho- IκB-α and anti-phospho-p65 antibodies were from Cell Signaling Technology(Beverly, MA, USA); anti-LC3 (L7543), anti-actin(A2066) antibodies, bafilomycin A1 (Baf-A1; B1793), 3-methyladenine (3-MA; M9281), wortmannin (WM; W1628), tert-butyl hydroperoxide (t-BHP; 458139), chloroquine (CQ; C6628), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 70-25-7), bacterial LPS (from Escherichia coli, serotype 0127:B8) and Human pLKO.1 lentiviral contructs from Open Biosystem, targeted Atg5 specific shRNA (NM_004849) were from Sigma-Aldrich (St. Louis, MO, USA). A NF-κB-luc adenoviral vector (Ad5HSV- NF-κB-luc) was a gift from K.C. Sohn (Chungnam National University, Daejeon, Korea). Recombinant mouse TNFα was from R&D Systems (Minneapolis, MN, USA). The caspase inhibitor (z-VAD-FMK; 627610), N-acetyl-L-cysteine (NAC; 106425) and diphenyene iodonium (DPI) were from Calbiochem (San Diego, CA, USA). Mito-TEMPO [(2-(2,2,6,6 tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride, monohydrate] (ALX-430–150-M005) and recombinant Fas Ligand (ALX-522-020-C005) were from Enzo Life Sciences (East Farmingdale, NY, USA);

C57BL/6J (wild-type: WT) mice were purchased from CLEA Japan Inc. (Tokyo, Japan). B6;129S-Tnfrsf1a^tm1Imx (p55, TNFR1-deficient) Tnfrsf1b^tm1Imx/J (p75, TNFR2-deficient) (TNFRsKO) mice and C57BL/6-Tg(Dmp1-Topaz)1lkal/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The protocols for all animal procedures were performed in accordance with Tohoku University regulations.
Recombinant mouse TNF-α for in vivo experiments was prepared in our laboratory as previously described [10 (link)]. Recombinant mouse TNF-α and recombinant human sclerostin (rhSCL) for in vitro experiments were purchased from R&D Systems (Minneapolis, MN, USA).

In a transwell co-culture system, RAW 264.7 cells (2×10⁵, 4×10⁵, 8×10⁵) seeded on a 0.4 μm Transwell insert (Millipore) were co-cultured with podocytes (4×10⁵) for 48 h in the absence or presence of 25 mM high glucose treatment. The ratios of podocytes (P) to macrophages (M) were 2:1, 2:2 and 2:4, respectively.
In the CM experiments, podocytes (4×10⁵) planted on six well plates were cultured overnight in normal RPMI 1640 media. Then, the cells were washed with PBS three times. After that, normal PRMI 1640 media (control), NC-CM or HG-CMwas added to podocytes for 24 - 72 h. In some experiments, 10μg/ml anti-TNF-α neutralizing antibody (RD, USA), 10μg/ml IgG1 Isotype control (RD, USA), 300μM ROS inhibitors (Tempo, sigma) or 10μM p38MAPK inhibitor (SB203580, RD, USA) was respectively added to cells with CM for 72 h. Besides, 10ng/ml recombinant mouse TNF-α (RD, USA) alone was applied to incubate podocytes for 72 h when necessary.

Anti-phospho p65 (Ser-536), Akt (Ser-473), JNK (Thr183/Tyr185), ERK (Thr-202, Tyr-204), p38 (Thr-180/Tyr-182), PARP (46D11) and caspase-3 (8G10) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Akt (C-20), p38 (C-20), JNK (FL), ERK1 (C-16), p65 (C-20-G), IκBα (L35A5), IRF8 (C-19) and β-actin (C-11) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). CXCL16 antibody and recombinant mouse TNF-α were obtained from R&D Systems (Minneapolis, MN, USA). Mouse TNF-α neutralizing antibody and 2-chloroadenosine were purchased from eBioscience (San Diego, CA, USA) and Sigma (St Louis, MO, USA), respectively.

Stereotaxic injections were performed by Washington University's animal surgery core using a David Koft stereotaxic frame. Adult male and female Cxcr7^GFP/+ animals were shaved and anesthetized with 2% isoflurane prior to surgery. The surgical site was cleaned with 75% ethanol, followed by 1% betadine. A 1.0 cm sagittal incision was made at the level of L2/L3 vertebrae for spinal cord injections or at the brain stem. A glass needle was inserted at the incision site using the Drummond Nanoject2. Stereotaxic injections were performed at a depth of 0.5 mm, just lateral to midline. A volume of 0.25 μl recombinant mouse TNFα (25 ng, R&D Systems), recombinant mouse IFNγ (25 ng, Biolegend), or vehicle (PBS) was injected. After microinjection, the dorsal muscle was sutured with 4–0 silk. The incision skin was pulled together using forceps and sutured using 4.0 nylon. The mice were kept on a heating pad until awake. Animals were sacrificed 72 hr postinjection.

A murine mesangial cell line, Mes13, was obtained from American Type Culture Collection (ATCC, Manassas, VA). Mes13 cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM) containing high glucose/Nutrient Mixture F-12 Ham supplemented with 10% fetal bovine serum (FBS). Human umbilical vein endothelial cells (HUVEC) were cultured with EGM^TM-2 BulletKit^TM (Lonza, Basel, Switzerland). An adherent macrophage cell line, Raw 264.7, was obtained from ATCC and were cultured with DMEM containing high glucose concentration with 10% FBS at 37 °C under 5% CO₂. For the analysis for M1/M2 macrophage subtype transition by CTGF or TNF-α, RAW264.7 cells were stimulated with vehicle or 1000 ng/ml CTGF (PROSPEC, East Brunswick, NJ) in DMEM without FBS, and then harvested 3 h after stimulation. Stimulation with 10 ng/ml recombinant mouse TNF-α (R&D systems, Minneapolis, MA) was performed at the same protocol, and collected at 3 h. To examine mRNA expression of Ccl2 in mesangial cells, Mes 13 cell were stimulated with 1 ng/ml TGF-β1 and/or 500 ng/ml CTGF, and then harvested 3 h after stimulation.