Gelatin

gelatinolytic activity of MMP9 and MMP2 upon MYCN inhibition in Y79 and LRB1 cells was determined by substrate gelatin zymography. Equal amount of proteins were separated on 10% SDS-PAGE gels containing 0.1% gelatin (Amresco, Solon, OH, USA). The gels were washed thrice for 15 min each in 2.5% Triton X-100 washing buffer and then incubated in incubation buffer containing 50 mM Tris-HCl, 10 mM CaCl₂, 1 M ZnCl₂ and 200 mM NaCl, pH 7.5 at 37 °C for 18 to 20 h. Subsequently, gels were stained with Coomassie solution (0.05% Coomassie brilliant blue R-250, in 40% methanol and 10% acetic acid) and destained with destaining solution (20% methanol and 10% acetic acid) to visualize the clear zones of gelatinolytic activity of MMPs against the blue background.

Standard protocol for the indirect ELISA was conducted by the procedure described previously [18 (link)]. Briefly, purified recombinant p30 diluted in carbonate buffer was coated on 96-well plates (Corning, NY, USA) (0.5 μg/mL, 100 μL/well) and incubated overnight at 4 °C. The plate was washed three times with PBST (0.05% Tween in 0.01 M PBS) and blocked with 1% gelatin (Amresco, Pittsburgh, PA, USA) in PBST for 2 h at 37 °C, then washed three times with PBST. Supernatants (50 μL) were added and incubated for 30 min at 37 °C. As a control, p30-immunized mice and unimmunized mice sera were diluted 1:100 separately in duplicate. After incubation, the plate was washed as described beforehand. Horseradish peroxidase (HRP) conjugated goat anti-mouse IgG + IgM + IgA (Bethyl, Montgomery, TX, USA) diluted 1:20,000 in PBST was dispensed into plates at 100 μL/well, and incubated for 30 min at 37 °C. Following washing three times with PBST, 100 μL chromogenic substrate solution (TMB) (Huzhou InnoReagents Corp., Zhejiang, China) was added into each well. Incubation took place at room temperature for 10 min, followed by the addition of 50 μL of 2 M sulfuric acid per well. The result was read at OD450 nm absorbance by using a microplate reader (Bio Tek, Winooski, VT, USA).

Procyanidin was purchased from Aladdin Co. Ltd. (Shanghai, China). Secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Toll-like receptor 4 (TLR-4) inhibitor TAK-242, phosphatidylinositol 3-kinase (PI3K) inhibitor AS-605240, and protein kinase B (Akt) inhibitor GSK690693 were purchased from Selleck Chemicals (Shanghai, China). RAGE antibody 553030 was purchased from Calbiochem (Merck, Darmstadt, Germany). Anti-HMGB1 polyclonal antibody 326052233 was purchased from SHINO-TEST Corporation (Sagamihara City, Japan) and GAPDH was purchased from Proteintech, Wuhan, China. Gelatin was purchased from Amresco (Solon, OH, USA). Zymogram renaturing buffer and developing buffer were purchased from Novex (Carlsbad, CA, USA). FBS was purchased from Gibco (Grand Island, NY, USA), and other cell culture media and supplements were purchased from HyClone (Logan, UT, USA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Antibody for β-actin was purchased from Sigma (St. Louis, MO). Antibodies for hypoxia inducible factor-1α (HIF-1α), heatshockprotein70 (HSP70), phosphorylated p38 (Tyr182) were purchased from Cell Signaling Technology (Beverly, MA). TF was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA.). Secondary antibodies were purchased from Sigma (St. Louis, MO). L-OHP was purchased from Jiangsu heng rui medicine (Lianyungang, China). Gelatin was purchased from Amresco (Solon, OH, USA). Zymogram renaturing buffer and Zymogram developing buffer were purchased from Novex (Carlsbad, CA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Gibco, and other cell culture media and supplements were purchased from HyClone (Logan, UT, USA). Coomassie brilliant blue G250 was purchased from Beyotime (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma Chemical Co (St. Louis, MO). All other chemicals were purchased from Sigma Chemical Co (St. Louis, MO). Hirudin was purchased from Genscript (Nanjing, China). p38 inhibitor SB 203580 was purchased from MedChemExpress (NJ, USA).

HA (M_w = 340 kDa) was purchased from Shandong Furuda Biotechnology Company (Linyi, China). Gelatin (from cowhide) is purchased from Amresco (USA). Methacrylate anhydride (MA, 94%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 98.5%), and N-hydroxysuccinimide (NHS, 99%) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China). DA, phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP, 98.0%) and sodium periodate were purchased from Sigma-Aldrich (St. Louis, MO, USA). N, N-dimethylformamide (DMF, 99.5%) and hyaluronidase (563 unit/mg) were obtained from Aladdin (Shanghai, China).